Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics

ABSTRACT

The invention provides isolated polypeptide and nucleic acid sequences derived from  Streptococcus pneumoniae  that are useful in diagnosis and therapy of pathological conditions; antibodies against the polypeptides; and methods for the production of the polypeptides. The invention also provides methods for the detection, prevention and treatment of pathological conditions resulting from bacterial infection.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/027,892, filed Dec. 30, 2004, which is a continuation of U.S.application Ser. No. 10/640,833, filed Aug. 14, 2003, which is acontinuation of U.S. application Ser. No. 09/583,110 (now U.S. Pat. No.6,699,703) filed May 26, 2000, which is a continuation-in-part of U.S.application Ser. No. 09/107,433 (now U.S. Pat. No. 6,800,744), filedJun. 30, 1998, which claims the benefit of U.S. Application No.60/085,131, filed May 12, 1998 and of U.S. Application No. 60/051,553,filed Jul. 2, 1997. The entire teachings of the above applications areincorporated herein by reference.

INCORPORATION BY REFERENCE OF MATERIAL ON COMPACT DISK

This application incorporates by reference the Sequence Listingcontained on the two compact disks (Copy 1 and Copy 2), filedconcurrently herewith, containing the following file:

File name: 3687.1000-032SequenceList.txt; created Sep. 16, 2006, 8,135KB in size.

This application also incorporates by reference Table 2 contained on thetwo compact disks (Copy 1 and Copy 2), filed concurrently herewith,containing the following file:

File name: Table2_(—)2.txt; created Aug. 21, 2006, 351 KB in size.

FIELD OF THE INVENTION

The invention relates to isolated nucleic acids and polypeptides derivedfrom Streptococcus pneumoniae that are useful as molecular targets fordiagnostics, prophylaxis and treatment of pathological conditions, aswell as materials and methods for the diagnosis, prevention, andamelioration of pathological conditions resulting from bacterialinfection.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae (S. pneumoniae) is a common, spherical,gram-positive bacterium. Worldwide it is a leading cause of illnessamong children, the elderly, and individuals with debilitating medicalconditions (Breiman, R. F. et al., 1994, JAMA 271: 1831). S. pneumoniaeis estimated to be the causal agent in 3,000 cases of meningitis, 50,000cases of bacteremia, 500,000 cases of pneumonia, and 7,000,000 cases ofotitis media annually in the United States alone (Reichler, M. R. etal., 1992, J. Infect. Dis. 166: 1346; Stool, S. E. and Field, M. J.,1989 Pediatr. Infect. Dis J. 8: S11). In the United States alone, 40,000deaths result annually from S. pneumoniae infections (Williams, W. W. etal., 1988 Ann. Intern. Med. 108: 616) with a death rate approaching 30%from bacteremia (Butler, J. C. et al., 1993, JAMA 270: 1826).Pneumococcal pneumonia is a serious problem among the elderly ofindustrialized nations (Käyhty, H. and Eskola, J., 1996 Emerg. Infect.Dis. 2: 289) and is a leading cause of death among children indeveloping nations (Käyhty, H. and Eskola, J., 1996 Emerg. Infect. Dis.2: 289; Stansfield, S. K., 1987 Pediatr. Infect. Dis. 6: 622).

Vaccines against S. pneumoniae have been available for a number ofyears. There are a large number of serotypes based on the polysaccharidecapsule (van Dam, J. E., Fleer, A., and Snippe, H., 1990 Antonie vanLeeuwenhoek 58: 1) although only a fraction of the serotypes seem to beassociated with infections (Martin, D. R. and Brett, M. S., 1996 N. Z.Med. J. 109: 288). A multivalent vaccine against capsularpolysaccharides of 23 serotypes (Smart, L. E., Dougall, A. J. andGridwood, R. W., 1987 J. Infect. 14: 209) has provided protection forsome groups but not for several groups at risk for pneumococcalinfections, such as infants and the elderly (Mäkel, P. H. et al., 1980Lancet 2: 547; Sankilampi, U., 1996 J. Infect. Dis. 173: 387).Conjugated pneumococcal capsular polysaccharide vaccines have somewhatimproved efficacy, but are costly and, therefore, are not likely to bein widespread use (Käyhty, H. and Eskola, J., 1996 Emerg. Infect. Dis.2: 289).

At one time, S. pneumoniae strains were uniformly susceptible topenicillin. The report of a penicillin-resistant strain of (Hansman, D.and Bullen, M. M., 1967 Lancet 1: 264) was followed rapidly by manyreports indicating the worldwide emergence of penicillin-resistant andpenicillin non-susceptible strains (Klugman, K. P., 1990 Clin.Microbiol. Rev. 3: 171). S. pneumoniae strains which are resistant tomultiple antibiotics (including penicillin) have also been observedrecently within the United States (Welby, P. L., 1994 Pediatr. Infect.Dis. J. 13: 281; Ducin, J. S. et al., 1995 Pediatr. Infect. Dis. J. 14:745; Butler, J. C., 1996 J. Infect. Dis. 174: 986) as well asinternationally (Boswell, T. C. et al., 1996; J. Infect. 33: 17;Catchpole, C., Fraise, A., and Wise, R., 1996 Microb. Drug Resist. 2:431; Tarasi, A. et al., 1997 Microb. Drug Resist. 3: 105).

A high incidence of morbidity is associated with invasive S. pneumoniaeinfections (Williams, W. W. et al., 1988 Ann. Intern. Med. 108: 616).Because of the incomplete effectiveness of currently available vaccinesand antibiotics, the identification of new targets for antimicrobialtherapies, including, but not limited to, the design of vaccines andantibiotics, which may help prevent infection or that may be useful infighting existing infections, is highly desirable.

SUMMARY OF THE INVENTION

The present invention fulfills the need for diagnostic tools andtherapeutics by providing bacterial-specific compositions and methodsfor detecting, treating, and preventing bacterial infection, inparticular S. pneumoniae infection.

The present invention encompasses isolated polypeptides and nucleicacids derived from S. pneumoniae that are useful as reagents fordiagnosis of bacterial infection, components of effective antibacterialvaccines, and/or as targets for antibacterial drugs, including anti-S.pneumoniae drugs. The nucleic acids and peptides of the presentinvention also have utility for diagnostics and therapeutics for S.pneumoniae and other Streptococcus species. They can also be used todetect the presence of S. pneumoniae and other Streptococcus species ina sample; and in screening compounds for the ability to interfere withthe S. pneumoniae life cycle or to inhibit S. pneumoniae infection. Morespecifically, this invention features compositions of nucleic acidscorresponding to entire coding sequences of S. pneumoniae proteins,including surface or secreted proteins or parts thereof, nucleic acidscapable of binding mRNA from S. pneumoniae proteins to block proteintranslation, and methods for producing S. pneumoniae proteins or partsthereof using peptide synthesis and recombinant DNA techniques. Thisinvention also features antibodies and nucleic acids useful as probes todetect S. pneumoniae infection. In addition, vaccine compositions andmethods for the protection or treatment of infection by S. pneumoniaeare within the scope of this invention.

The nucleotide sequences provided in SEQ ID NO: 1-SEQ ID NO: 2661, afragment thereof, or a nucleotide sequence at least 99.5% identical to asequence contained within SEQ ID NO: 1-SEQ ID NO: 2661 may be “provided”in a variety of medias to facilitate use thereof. As used herein,“provided” refers to a manufacture, other than an isolated nucleic acidmolecule, which contains a nucleotide sequence of the present invention,i.e., the nucleotide sequence provided in SEQ ID NO: 1-SEQ ID NO: 2661,a fragment thereof, or a nucleotide sequence at least 99.5% identical toa sequence contained within SEQ ID NO: 1-SEQ ID NO: 2661. Uses for andmethods for providing nucleotide sequences in a variety of media is wellknown in the art (see e.g., EPO Publication No. EP 0 756 006)

In one application of this embodiment, a nucleotide sequence of thepresent invention can be recorded on computer readable media. As usedherein, “computer readable media” refers to any media which can be readand accessed directly by a computer. Such media include, but are notlimited to: magnetic storage media, such as floppy discs, hard discstorage media, and magnetic tape; optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; and hybrids of thesecategories such as magnetic/optical storage media. A person skilled inthe art can readily appreciate how any of the presently known computerreadable media can be used to create a manufacture comprising computerreadable media having recorded thereon a nucleotide sequence of thepresent invention.

As used herein, “recorded” refers to a process for storing informationon computer readable media. A person skilled in the art can readilyadopt any of the presently known methods for recording information oncomputer readable media to generate manufactures comprising thenucleotide sequence information of the present invention.

A variety of data storage structures are available to a person skilledin the art for creating a computer readable media having recordedthereon a nucleotide sequence of the present invention. The choice ofthe data storage structure will generally be based on the means chosento access the stored information. In addition, a variety of dataprocessor programs and formats can be used to store the nucleotidesequence information of the present invention on computer readablemedia. The sequence information can be represented in a word processingtext file, formatted in commercially-available software such asWordPerfect and Microsoft Word, or represented in the form of an ASCIIfile, stored in a database application, such as DB2, Sybase, Oracle, orthe like. A person skilled in the art can readily adapt any number ofdata processor structuring formats (e.g. text file or database) in orderto obtain computer readable media having recorded thereon the nucleotidesequence information of the present invention.

By providing the nucleotide sequence of SEQ ID NO: 1-SEQ ID NO: 2661, afragment thereof, or a nucleotide sequence at least 99.5% identical to asequence contained within SEQ ID NO: 1-SEQ ID NO: 2661 in computerreadable form, a person skilled in the art can routinely access thesequence information for a variety of purposes. Computer software ispublicly available which allows a person skilled in the art to accesssequence information provided in a computer readable media. Examples ofsuch computer software include programs of the “Staden Package”, “DNAStar”, “MacVector”, GCG “Wisconsin Package” (Genetics Computer Group,Madison, Wis.) and “NCBI toolbox” (National Center for BiotechnologyInformation).

Computer algorithms enable the identification of S. pneumoniae openreading frames (ORFs) within SEQ ID NO: 1-SEQ ID NO: 2661 which containhomology to ORFs or proteins from other organisms. Examples of suchsimilarity-search algorithms include the BLAST [Altschul et al., J. Mol.Biol. 215:403-410 (1990)] and Smith-Waterman [Smith and Waterman (1981)Advances in Applied Mathematics, 2:482-489] search algorithms. Thesealgorithms are utilized on computer systems as exemplified below. TheORFs so identified represent protein encoding fragments within the S.pneumoniae genome and are useful in producing commercially importantproteins such as enzymes used in fermentation reactions and in theproduction of commercially useful metabolites.

The present invention further provides systems, particularlycomputer-based systems, which contain the sequence information describedherein. Such systems are designed to identify commercially importantfragments of the S. pneumoniae genome. As used herein, “a computer-basedsystem” refers to the hardware means, software means, and data storagemeans used to analyze the nucleotide sequence information of the presentinvention. The minimum hardware means of the computer-based systems ofthe present invention comprises a central processing unit (CPU), inputmeans, output means, and data storage means. A person skilled in the artcan readily appreciate that any one of the currently availablecomputer-based systems is suitable for use in the present invention. Thecomputer-based systems of the present invention comprise a data storagemeans having stored therein a nucleotide sequence of the presentinvention and the necessary hardware means and software means forsupporting and implementing a search means. As used herein, “datastorage means” refers to memory which can store nucleotide sequenceinformation of the present invention, or a memory access means which canaccess manufactures having recorded thereon the nucleotide sequenceinformation of the present invention.

As used herein, “search means” refers to one or more programs which areimplemented on the computer-based system to compare a target sequence ortarget structural motif with the sequence information stored within thedata storage means. Search means are used to identify fragments orregions of the S. pneumoniae genome which are similar to, or “match”, aparticular target sequence or target motif. A variety of knownalgorithms are known in the art and have been disclosed publicly, and avariety of commercially available software for conducting homology-basedsimilarity searches are available and can be used in the computer-basedsystems of the present invention. Examples of such software include, butis not limited to, FASTA (GCG Wisconsin Package), Bic_SW (CompugenBioccelerator, BLASTN2, BLASTP2 and BLASTX2 (NCBI) and Motifs (GCG).BLASTN2, A person skilled in the art can readily recognize that any oneof the available algorithms or implementing software packages forconducting homology searches can be adapted for use in the presentcomputer-based systems.

As used herein, a “target sequence” can be any DNA or amino acidsequence of six or more nucleotides or two or more amino acids. A personskilled in the art can readily recognize that the longer a targetsequence is, the less likely a target sequence will be present as arandom occurrence in the database. The most preferred sequence length ofa target sequence is from about 10 to 100 amino acids or from about 30to 300 nucleotide residues. However, it is well recognized that manygenes are longer than 500 amino acids, or 1.5 kb in length, and thatcommercially important fragments of the S. pneumoniae genome, such assequence fragments involved in gene expression and protein processing,will often be shorter than 30 nucleotides.

As used herein, “a target structural motif,” or “target motif,” refersto any rationally selected sequence or combination of sequences in whichthe sequence(s) are chosen based on a specific functional domain orthree-dimensional configuration which is formed upon the folding of thetarget polypeptide. There are a variety of target motifs known in theart. Protein target motifs include, but are not limited to, enzymaticactive sites, membrane spanning regions, and signal sequences. Nucleicacid target motifs include, but are not limited to, promoter sequences,hairpin structures and inducible expression elements (protein bindingsequences).

A variety of structural formats for the input and output means can beused to input and output the information in the computer-based systemsof the present invention. A preferred format for an output means ranksfragments of the S. pneumoniae genome possessing varying degrees ofhomology to the target sequence or target motif. Such presentationprovides a person skilled in the art with a ranking of sequences whichcontain various amounts of the target sequence or target motif andidentifies the degree of homology contained in the identified fragment.

A variety of comparing means can be used to compare a target sequence ortarget motif with the data storage means to identify sequence fragmentsof the S. pneumoniae genome. In the present examples, implementingsoftware which implement the BLASTP2 and bic_SW algorithms (Altschul etal., J. Mol. Biol. 215:403-410 (1990); Compugen Biocellerator) was usedto identify open reading frames within the S. pneumoniae genome. Aperson skilled in the art can readily recognize that any one of thepublicly available homology search programs can be used as the searchmeans for the computer-based systems of the present invention.

The invention features S. pneumoniae polypeptides, preferably asubstantially pure preparation of an S. pneumoniae polypeptide, or arecombinant S. pneumoniae polypeptide. In preferred embodiments: thepolypeptide has biological activity; the polypeptide has an amino acidsequence at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical to anamino acid sequence of the invention contained in the Sequence Listing,preferably it has about 65% sequence identity with an amino acidsequence of the invention contained in the Sequence Listing, and mostpreferably it has about 92% to about 99% sequence identity with an aminoacid sequence of the invention contained in the Sequence Listing; thepolypeptide has an amino acid sequence essentially the same as an aminoacid sequence of the invention contained in the Sequence Listing; thepolypeptide is at least 5, 10, 20, 50, 100, or 150 amino acid residuesin length; the polypeptide includes at least 5, preferably at least 10,more preferably at least 20, more preferably at least 50, 100, or 150contiguous amino acid residues of the invention contained in theSequence Listing. In yet another preferred embodiment, the amino acidsequence which differs in sequence identity by about 7% to about 8% fromthe S. pneumoniae amino acid sequences of the invention contained in theSequence Listing is also encompassed by the invention.

In preferred embodiments: the S. pneumoniae polypeptide is encoded by anucleic acid of the invention contained in the Sequence Listing, or by anucleic acid having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99%homology with a nucleic acid of the invention contained in the SequenceListing.

In a preferred embodiment, the subject S. pneumoniae polypeptide differsin amino acid sequence at 1, 2, 3, 5, 10 or more residues from asequence of the invention contained in the Sequence Listing. Thedifferences, however, are such that the S. pneumoniae polypeptideexhibits an S. pneumoniae biological activity, e.g., the S. pneumoniaepolypeptide retains a biological activity of a naturally occurring S.pneumoniae enzyme.

In preferred embodiments, the polypeptide includes all or a fragment ofan amino acid sequence of the invention contained in the SequenceListing; fused, in reading frame, to additional amino acid residues,preferably to residues encoded by genomic DNA 5′ or 3′ to the genomicDNA which encodes a sequence of the invention contained in the SequenceListing.

In yet other preferred embodiments, the S. pneumoniae polypeptide is arecombinant fusion protein having a first S. pneumoniae polypeptideportion and a second polypeptide portion, e.g., a second polypeptideportion having an amino acid sequence unrelated to S. pneumoniae. Thesecond polypeptide portion can be, e.g., any ofglutathione-S-transferase, a DNA binding domain, or a polymeraseactivating domain. In preferred embodiment the fusion protein can beused in a two-hybrid assay.

Polypeptides of the invention include those which arise as a result ofalternative transcription events, alternative RNA splicing events, andalternative translational and posttranslational events.

In a preferred embodiment, the encoded S. pneumoniae polypeptide differs(e.g., by amino acid substitution, addition or deletion of at least oneamino acid residue) in amino acid sequence at 1, 2, 3, 5, 10 or moreresidues, from a sequence of the invention contained in the SequenceListing. The differences, however, are such that: the S. pneumoniaeencoded polypeptide exhibits a S. pneumoniae biological activity, e.g.,the encoded S. pneumoniae enzyme retains a biological activity of anaturally occurring S. pneumoniae.

In preferred embodiments, the encoded polypeptide includes all or afragment of an amino acid sequence of the invention contained in theSequence Listing; fused, in reading frame, to additional amino acidresidues, preferably to residues encoded by genomic DNA 5′ or 3′ to thegenomic DNA which encodes a sequence of the invention contained in theSequence Listing.

The S. pneumoniae strain, 14453, from which genomic sequences have beensequenced, has been deposited on Jun. 26, 1997 in the American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209,and assigned the ATCC designation # 55987.

Included in the invention are: allelic variations; natural mutants;induced mutants; proteins encoded by DNA that hybridize under high orlow stringency conditions to a nucleic acid which encodes a polypeptideof the invention contained in the Sequence Listing (for definitions ofhigh and low stringency see Current Protocols in Molecular Biology, JohnWiley & Sons, New York, 1989, 6.3.1-6.3.6, hereby incorporated byreference); and, polypeptides specifically bound by antisera to S.pneumoniae polypeptides, especially by antisera to an active site orbinding domain of S. pneumoniae polypeptide. The invention also includesfragments, preferably biologically active fragments. These and otherpolypeptides are also referred to herein as S. pneumoniae polypeptideanalogs or variants.

The invention further provides nucleic acids, e.g., RNA or DNA, encodinga polypeptide of the invention. This includes double stranded nucleicacids as well as coding and antisense single strands.

In preferred embodiments, the subject S. pneumoniae nucleic acid willinclude a transcriptional regulatory sequence, e.g. at least one of atranscriptional promoter or transcriptional enhancer sequence, operablylinked to the S. pneumoniae gene sequence, e.g., to render the S.pneumoniae gene sequence suitable for expression in a recombinant hostcell.

In yet a further preferred embodiment, the nucleic acid which encodes anS. pneumoniae polypeptide of the invention, hybridizes under stringentconditions to a nucleic acid probe corresponding to at least 8consecutive nucleotides of the invention contained in the SequenceListing; more preferably to at least 12 consecutive nucleotides of theinvention contained in the Sequence Listing; more preferably to at least20 consecutive nucleotides of the invention contained in the SequenceListing; more preferably to at least 40 consecutive nucleotides of theinvention contained in the Sequence Listing.

In another aspect, the invention provides a substantially pure nucleicacid having a nucleotide sequence which encodes an S. pneumoniaepolypeptide. In preferred embodiments: the encoded polypeptide hasbiological activity; the encoded polypeptide has an amino acid sequenceat least 60%, 70%, 80%, 90%, 95%, 98%, or 99% homologous to an aminoacid sequence of the invention contained in the Sequence Listing; theencoded polypeptide has an amino acid sequence essentially the same asan amino acid sequence of the invention contained in the SequenceListing; the encoded polypeptide is at least 5, 10, 20, 50, 100, or 150amino acids in length; the encoded polypeptide comprises at least 5,preferably at least 10, more preferably at least 20, more preferably atleast 50, 100, or 150 contiguous amino acids of the invention containedin the Sequence Listing.

In another aspect, the invention encompasses: a vector including anucleic acid which encodes an S. pneumoniae polypeptide or an S.pneumoniae polypeptide variant as described herein; a host celltransfected with the vector; and a method of producing a recombinant S.pneumoniae polypeptide or S. pneumoniae polypeptide variant; includingculturing the cell, e.g., in a cell culture medium, and isolating an S.pneumoniae polypeptide or an S. pneumoniae polypeptide variant, e.g.,from the cell or from the cell culture medium.

In another series of embodiments, the invention provides isolatednucleic acids comprising sequences at least about 8 nucleotides inlength, more preferably at least about 12 nucleotides in length, andmost preferably at least about 15-20 nucleotides in length, thatcorrespond to a subsequence of any one of SEQ ID NO: 1-SEQ ID NO: 2661or complements thereof. Alternatively, the nucleic acids comprisesequences contained within any ORF (open reading frame), including acomplete protein-coding sequence, of which any of SEQ ID NO: 1-SEQ IDNO: 2661 forms a part. The invention encompasses sequence-conservativevariants and function-conservative variants of these sequences. Thenucleic acids may be DNA, RNA, DNA/RNA duplexes, protein-nucleic acid(PNA), or derivatives thereof.

In another aspect, the invention features, a purified recombinantnucleic acid having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%homology with a sequence of the invention contained in the SequenceListing.

In another aspect, the invention features nucleic acids capable ofbinding mRNA of S. pneumoniae. Such nucleic acid is capable of acting asantisense nucleic acid to control the translation of mRNA of S.pneumoniae. A further aspect features a nucleic acid which is capable ofbinding specifically to an S. pneumoniae nucleic acid. These nucleicacids are also referred to herein as complements and have utility asprobes and as capture reagents.

In another aspect, the invention features an expression systemcomprising an open reading frame corresponding to S. pneumoniae nucleicacid. The nucleic acid further comprises a control sequence compatiblewith an intended host. The expression system is useful for makingpolypeptides corresponding to S. pneumoniae nucleic acid.

In another aspect, the invention features a cell transformed with theexpression system to produce S. pneumoniae polypeptides.

In yet another embodiment, the invention encompasses reagents fordetecting bacterial infection, including S. pneumoniae infection, whichcomprise at least one S. pneumoniae-derived nucleic acid defined by anyone of SEQ ID NO: 1-SEQ ID NO: 2661, or sequence-conservative orfunction-conservative variants thereof. Alternatively, the diagnosticreagents comprise polypeptide sequences that are contained within anyopen reading frames (ORFs), including complete protein-coding sequences,contained within any of SEQ ID NO: 1-SEQ ID NO: 2661, or polypeptidesequences contained within any of SEQ ID NO: 2662-SEQ ID NO: 5322, orpolypeptides of which any of the above sequences forms a part, orantibodies directed against any of the above peptide sequences orfunction-conservative variants and/or fragments thereof.

The invention further provides antibodies, preferably monoclonalantibodies, which specifically bind to the polypeptides of theinvention. Methods are also provided for producing antibodies in a hostanimal. The methods of the invention comprise immunizing an animal withat least one S. pneumoniae-derived immunogenic component, wherein theimmunogenic component comprises one or more of the polypeptides encodedby any one of SEQ ID NO: 1-SEQ ID NO: 2661 or sequence-conservative orfunction-conservative variants thereof; or polypeptides that arecontained within any ORFs, including complete protein-coding sequences,of which any of SEQ ID NO: 1-SEQ ID NO: 2661 forms a part; orpolypeptide sequences contained within any of SEQ ID NO: 2662-SEQ ID NO:5322; or polypeptides of which any of SEQ ID NO: 2662-SEQ ID NO: 5322forms a part. Host animals include any warm blooded animal, includingwithout limitation mammals and birds. Such antibodies have utility asreagents for immunoassays to evaluate the abundance and distribution ofS. pneumoniae-specific antigens.

In yet another aspect, the invention provides a method for detectingbacterial antigenic components in a sample, which comprises the stepsof: (i) contacting a sample suspected to contain a bacterial antigeniccomponent with a bacterial-specific antibody, under conditions in whicha stable antigen-antibody complex can form between the antibody andbacterial antigenic components in the sample; and (ii) detecting anyantigen-antibody complex formed in step (i), wherein detection of anantigen-antibody complex indicates the presence of at least onebacterial antigenic component in the sample. In different embodiments ofthis method, the antibodies used are directed against a sequence encodedby any of SEQ ID NO: 1-SEQ ID NO: 2661 or sequence-conservative orfunction-conservative variants thereof, or against a polypeptidesequence contained in any of SEQ ID NO: 2662-SEQ ID NO: 5322 orfunction-conservative variants thereof.

In yet another aspect, the invention provides a method for detectingantibacterial-specific antibodies in a sample, which comprises: (i)contacting a sample suspected to contain antibacterial-specificantibodies with a S. pneumoniae antigenic component, under conditions inwhich a stable antigen-antibody complex can form between the S.pneumoniae antigenic component and antibacterial antibodies in thesample; and (ii) detecting any antigen-antibody complex formed in step(i), wherein detection of an antigen-antibody complex indicates thepresence of antibacterial antibodies in the sample. In differentembodiments of this method, the antigenic component is encoded by asequence contained in any of SEQ ID NO: 1-SEQ ID NO: 2661 orsequence-conservative and function-conservative variants thereof, or isa polypeptide sequence contained in any of SEQ ID NO: 2662-SEQ ID NO:5322 or function-conservative variants thereof.

In another aspect, the invention features a method of generatingvaccines for immunizing an individual against S. pneumoniae. The methodincludes: immunizing a subject with an S. pneumoniae polypeptide, e.g.,a surface or secreted polypeptide, or active portion thereof, and apharmaceutically acceptable carrier. Such vaccines have therapeutic andprophylactic utilities.

In another aspect, the invention features a method of evaluating acompound, e.g. a polypeptide, e.g., a fragment of a host cellpolypeptide, for the ability to bind an S. pneumoniae polypeptide. Themethod includes: contacting the candidate compound with an S. pneumoniaepolypeptide and determining if the compound binds or otherwise interactswith an S. pneumoniae polypeptide. Compounds which bind S. pneumoniaeare candidates as activators or inhibitors of the bacterial life cycle.These assays can be performed in vitro or in vivo.

In another aspect, the invention features a method of evaluating acompound, e.g. a polypeptide, e.g., a fragment of a host cellpolypeptide, for the ability to bind an S. pneumoniae nucleic acid,e.g., DNA or RNA. The method includes: contacting the candidate compoundwith an S. pneumoniae nucleic acid and determining if the compound bindsor otherwise interacts with an S. pneumoniae polypeptide. Compoundswhich bind S. pneumoniae are candidates as activators or inhibitors ofthe bacterial life cycle. These assays can be performed in vitro or invivo.

DETAILED DESCRIPTION OF THE INVENTION

The sequences of the present invention include the specific nucleic acidand amino acid sequences set forth in the Sequence Listing that forms apart of the present specification, and which are designated SEQ ID NO:1-SEQ ID NO: 5322. Use of the terms “SEQ ID NO: 1-SEQ ID NO: 2661”, “SEQID NO: 2662-SEQ ID NO: 5322”, “the sequences depicted in Table 2”, etc.,is intended, for convenience, to refer to each individual SEQ ID NOindividually, and is not intended to refer to the genus of thesesequences. In other words, it is a shorthand for listing all of thesesequences individually. The invention encompasses each sequenceindividually, as well as any combination thereof.

DEFINITIONS

“Nucleic acid” or “polynucleotide” as used herein refers to purine- andpyrimidine-containing polymers of any length, either polyribonucleotidesor polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides.This includes single- and double-stranded molecules, i.e., DNA-DNA,DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNA)formed by conjugating bases to an amino acid backbone. This alsoincludes nucleic acids containing modified bases.

A nucleic acid or polypeptide sequence that is “derived from” adesignated sequence refers to a sequence that corresponds to a region ofthe designated sequence. For nucleic acid sequences, this encompassessequences that are homologous or complementary to the sequence, as wellas “sequence-conservative variants” and “function-conservativevariants.” For polypeptide sequences, this encompasses“function-conservative variants.” Sequence-conservative variants arethose in which a change of one or more nucleotides in a given codonposition results in no alteration in the amino acid encoded at thatposition. Function-conservative variants are those in which a givenamino acid residue in a polypeptide has been changed without alteringthe overall conformation and function of the native polypeptide,including, but not limited to, replacement of an amino acid with onehaving similar physico-chemical properties (such as, for example,acidic, basic, hydrophobic, and the like).“Function-conservative”variants also include any polypeptides that havethe ability to elicit antibodies specific to a designated polypeptide.

An “S. pneumoniae-derived” nucleic acid or polypeptide sequence may ormay not be present in other bacterial species, and may or may not bepresent in all S. pneumoniae strains. This term is intended to refer tothe source from which the sequence was originally isolated. Thus, a S.pneumoniae-derived polypeptide, as used herein, may be used, e.g., as atarget to screen for a broad spectrum antibacterial agent, to search forhomologous proteins in other species of bacteria or in eukaryoticorganisms such as fungi and humans, etc.

A purified or isolated polypeptide or a substantially pure preparationof a polypeptide are used interchangeably herein and, as used herein,mean a polypeptide that has been separated from other proteins, lipids,and nucleic acids with which it naturally occurs. Preferably, thepolypeptide is also separated from substances, e.g., antibodies or gelmatrix, e.g., polyacrylamide, which are used to purify it. Preferably,the polypeptide constitutes at least 10, 20, 50 70, 80 or 95% dry weightof the purified preparation. Preferably, the preparation contains:sufficient polypeptide to allow protein sequencing; at least 1, 10, or100 mg of the polypeptide.

A purified preparation of cells refers to, in the case of plant oranimal cells, an in vitro preparation of cells and not an entire intactplant or animal. In the case of cultured cells or microbial cells, itconsists of a preparation of at least 10% and more preferably 50% of thesubject cells.

A purified or isolated or a substantially pure nucleic acid, e.g., asubstantially pure DNA, (are terms used interchangeably herein) is anucleic acid which is one or both of the following: not immediatelycontiguous with both of the coding sequences with which it isimmediately contiguous (i.e., one at the 5′ end and one at the 3′ end)in the naturally-occurring genome of the organism from which the nucleicacid is derived; or which is substantially free of a nucleic acid withwhich it occurs in the organism from which the nucleic acid is derived.The term includes, for example, a recombinant DNA which is incorporatedinto a vector, e.g., into an autonomously replicating plasmid or virus,or into the genomic DNA of a prokaryote or eukaryote, or which exists asa separate molecule (e.g., a cDNA or a genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of other DNAsequences. Substantially pure DNA also includes a recombinant DNA whichis part of a hybrid gene encoding additional S. pneumoniae DNA sequence.

A “contig” as used herein is a nucleic acid representing a continuousstretch of genomic sequence of an organism.

An “open reading frame”, also referred to herein as ORF, is a region ofnucleic acid which encodes a polypeptide. This region usually representsthe total coding region for the polypeptide and can be determined from astop to stop codon or from a start to stop codon.

As used herein, a “coding sequence” is a nucleic acid which istranscribed into messenger RNA and/or translated into a polypeptide whenplaced under the control of appropriate regulatory sequences. Theboundaries of the coding sequence are determined by a translation startcodon at the five prime terminus and a translation stop codon at thethree prime terminus. A coding sequence can include but is not limitedto messenger RNA, synthetic DNA, and recombinant nucleic acid sequences.

A “complement” of a nucleic acid as used herein refers to ananti-parallel or antisense sequence that participates in Watson-Crickbase-pairing with the original sequence.

A “gene product” is a protein or structural RNA which is specificallyencoded by a gene.

As used herein, the term “probe” refers to a nucleic acid, peptide orother chemical entity which specifically binds to a molecule ofinterest. Probes are often associated with or capable of associatingwith a label. A label is a chemical moiety capable of detection. Typicallabels comprise dyes, radioisotopes, luminescent and chemiluminescentmoieties, fluorophores, enzymes, precipitating agents, amplificationsequences, and the like. Similarly, a nucleic acid, peptide or otherchemical entity which specifically binds to a molecule of interest andimmobilizes such molecule is referred herein as a “capture ligand”.Capture ligands are typically associated with or capable of associatingwith a support such as nitro-cellulose, glass, nylon membranes, beads,particles and the like. The specificity of hybridization is dependent onconditions such as the base pair composition of the nucleotides, and thetemperature and salt concentration of the reaction. These conditions arereadily discernable to one of ordinary skill in the art using routineexperimentation.

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared×100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, acomparison is made when two sequences are aligned to give maximumhomology.

Nucleic acids are hybridizable to each other when at least one strand ofa nucleic acid can anneal to the other nucleic acid under definedstringency conditions. Stringency of hybridization is determined by: (a)the temperature at which hybridization and/or washing is performed; and(b) the ionic strength and polarity of the hybridization and washingsolutions. Hybridization requires that the two nucleic acids containcomplementary sequences; depending on the stringency of hybridization,however, mismatches may be tolerated. Typically, hybridization of twosequences at high stingency (such as, for example, in a solution of0.5×SSC, at 65° C.) requires that the sequences be essentiallycompletely homologous. Conditions of intermediate stringency (such as,for example, 2×SSC at 65° C.) and low stringency (such as, for example2×SSC at 55° C.), require correspondingly less overall complementaritybetween the hybridizing sequences. (1×SSC is 0.15 M NaCl, 0.015 M Nacitrate).

The terms peptides, proteins, and polypeptides are used interchangeablyherein.

As used herein, the term “surface protein” refers to all surfaceaccessible proteins, e.g. inner and outer membrane proteins, proteinsadhering to the cell wall, and secreted proteins.

A polypeptide has S. pneumoniae biological activity if it has one, twoand preferably more of the following properties: (1) if when expressedin the course of an S. pneumoniae infection, it can promote, or mediatethe attachment of S. pneumoniae to a cell; (2) it has an enzymaticactivity, structural or regulatory function characteristic of an S.pneumoniae protein; (3) or the gene which encodes it can rescue a lethalmutation in an S. pneumoniae gene. A polypeptide has biological activityif it is an antagonist, agonist, or super-agonist of a polypeptidehaving one of the above-listed properties.

A biologically active fragment or analog is one having an in vivo or invitro activity which is characteristic of the S. pneumoniae polypeptidesof the invention contained in the Sequence Listing, or of othernaturally occurring S. pneumoniae polypeptides, e.g., one or more of thebiological activities described herein. Especially preferred arefragments which exist in vivo, e.g., fragments which arise from posttranscriptional processing or which arise from translation ofalternatively spliced RNA's. Fragments include those expressed in nativeor endogenous cells as well as those made in expression systems, e.g.,in CHO cells. Because peptides such as S. pneumoniae polypeptides oftenexhibit a range of physiological properties and because such propertiesmay be attributable to different portions of the molecule, a useful S.pneumoniae fragment or S. pneumoniae analog is one which exhibits abiological activity in any biological assay for S. pneumoniae activity.Most preferably the fragment or analog possesses 10%, preferably 40%,more preferably 60%, 70%, 80% or 90% or greater of the activity of S.pneumoniae, in any in vivo or in vitro assay.

Analogs can differ from naturally occurring S. pneumoniae polypeptidesin amino acid sequence or in ways that do not involve sequence, or both.Non-sequence modifications include changes in acetylation, methylation,phosphorylation, carboxylation, or glycosylation. Preferred analogsinclude S. pneumoniae polypeptides (or biologically active fragmentsthereof) whose sequences differ from the wild-type sequence by one ormore conservative amino acid substitutions or by one or morenon-conservative amino acid substitutions, deletions, or insertionswhich do not substantially diminish the biological activity of the S.pneumoniae polypeptide. Conservative substitutions typically include thesubstitution of one amino acid for another with similar characteristics,e.g., substitutions within the following groups: valine, glycine;glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamicacid; asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. Other conservative substitutions can be made inview of the table below.

TABLE 1 CONSERVATIVE AMINO ACID REPLACEMENTS For Amino Acid Code Replacewith any of Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-Cys Arginine RD-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn,D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln AsparticAcid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys,S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu,D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln,D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, β-Ala, Acp Isoleucine I D-Ile,Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val, Leu,D-Leu, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met,D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile, D-Ile,Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His,D-His, Trp, D-Trp, Trans-3,4, or 5-phenylproline, cis-3,4, or5-phenylproline Proline P D-Pro, L-I-thioazolidine-4-carboxylic acid,D-or L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr,allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr,Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val TyrosineY D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile,D-Ile, Met, D-Met

Other analogs within the invention are those with modifications whichincrease peptide stability; such analogs may contain, for example, oneor more non-peptide bonds (which replace the peptide bonds) in thepeptide sequence. Also included are: analogs that include residues otherthan naturally occurring L-amino acids, e.g., D-amino acids ornon-naturally occurring or synthetic amino acids, e.g., β or γ aminoacids; and cyclic analogs.

As used herein, the term “fragment”, as applied to an S. pneumoniaeanalog, will ordinarily be at least about 20 residues, more typically atleast about 40 residues, preferably at least about 60 residues inlength. Fragments of S. pneumoniae polypeptides can be generated bymethods known to those skilled in the art. The ability of a candidatefragment to exhibit a biological activity of S. pneumoniae polypeptidecan be assessed by methods known to those skilled in the art asdescribed herein. Also included are S. pneumoniae polypeptidescontaining residues that are not required for biological activity of thepeptide or that result from alternative mRNA splicing or alternativeprotein processing events.

An “immunogenic component” as used herein is a moiety, such as an S.pneumoniae polypeptide, analog or fragment thereof, that is capable ofeliciting a humoral and/or cellular immune response in a host animal.

An “antigenic component” as used herein is a moiety, such as an S.pneumoniae polypeptide, analog or fragment thereof, that is capable ofbinding to a specific antibody with sufficiently high affinity to form adetectable antigen-antibody complex.

The term “antibody” as used herein is intended to include fragmentsthereof which are specifically reactive with S. pneumoniae polypeptides.

As used herein, the term “cell-specific promoter” means a DNA sequencethat serves as a promoter, i.e., regulates expression of a selected DNAsequence operably linked to the promoter, and which effects expressionof the selected DNA sequence in specific cells of a tissue. The termalso covers so-called “leaky” promoters, which regulate expression of aselected DNA primarily in one tissue, but cause expression in othertissues as well.

Misexpression, as used herein, refers to a non-wild type pattern of geneexpression. It includes: expression at non-wild type levels, i.e., overor under expression; a pattern of expression that differs from wild typein terms of the time or stage at which the gene is expressed, e.g.,increased or decreased expression (as compared with wild type) at apredetermined developmental period or stage; a pattern of expressionthat differs from wild type in terms of decreased expression (ascompared with wild type) in a predetermined cell type or tissue type; apattern of expression that differs from wild type in terms of thesplicing size, amino acid sequence, post-translational modification, orbiological activity of the expressed polypeptide; a pattern ofexpression that differs from wild type in terms of the effect of anenvironmental stimulus or extracellular stimulus on expression of thegene, e.g., a pattern of increased or decreased expression (as comparedwith wild type) in the presence of an increase or decrease in thestrength of the stimulus.

As used herein, “host cells” and other such terms denotingmicroorganisms or higher eukaryotic cell lines cultured as unicellularentities refers to cells which can become or have been used asrecipients for a recombinant vector or other transfer DNA, and includethe progeny of the original cell which has been transfected. It isunderstood by individuals skilled in the art that the progeny of asingle parental cell may not necessarily be completely identical ingenomic or total DNA compliment to the original parent, due to accidentor deliberate mutation.

As used herein, the term “control sequence” refers to a nucleic acidhaving a base sequence which is recognized by the host organism toeffect the expression of encoded sequences to which they are ligated.The nature of such control sequences differs depending upon the hostorganism; in prokaryotes, such control sequences generally include apromoter, ribosomal binding site, terminators, and in some casesoperators; in eukaryotes, generally such control sequences includepromoters, terminators and in some instances, enhancers. The termcontrol sequence is intended to include at a minimum, all componentswhose presence is necessary for expression, and may also includeadditional components whose presence is advantageous, for example,leader sequences.

As used herein, the term “operably linked” refers to sequences joined orligated to function in their intended manner. For example, a controlsequence is operably linked to coding sequence by ligation in such a waythat expression of the coding sequence is achieved under conditionscompatible with the control sequence and host cell.

The “metabolism” of a substance, as used herein, means any aspect of theexpression, function, action, or regulation of the substance. Themetabolism of a substance includes modifications, e.g., covalent ornon-covalent modifications of the substance. The metabolism of asubstance includes modifications, e.g., covalent or non-covalentmodification, the substance induces in other substances. The metabolismof a substance also includes changes in the distribution of thesubstance. The metabolism of a substance includes changes the substanceinduces in the distribution of other substances.

A “sample” as used herein refers to a biological sample, such as, forexample, tissue or fluid isolated from an individual (including withoutlimitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva andtissue sections) or from in vitro cell culture constituents, as well assamples from the environment.

Technical and scientific terms used herein have the meanings commonlyunderstood by one of ordinary skill in the art to which the presentinvention pertains, unless otherwise defined. Reference is made hereinto various methodologies known to those of skill in the art.Publications and other materials setting forth such known methodologiesto which reference is made are incorporated herein by reference in theirentireties as though set forth in full. The practice of the inventionwill employ, unless otherwise indicated, conventional techniques ofchemistry, molecular biology, microbiology, recombinant DNA, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. See e.g., Sambrook, Fritsch, andManiatis, Molecular Cloning; Laboratory Manual 2nd ed. (1989); DNACloning, Volumes I and II (D. N Glover ed. 1985); OligonucleotideSynthesis (M. J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames& S. J. Higgins eds. 1984); the series, Methods in Enzymology (AcademicPress, Inc.), particularly Vol. 154 and Vol. 155 (Wu and Grossman,eds.); PCR—A Practical Approach (McPherson, Quirke, and Taylor, eds.,1991); Immunology, 2d Edition, 1989, Roitt et al., C. V. Mosby Company,and New York; Advanced Immunology, 2d Edition, 1991, Male et al., GrowerMedical Publishing, New York.; DNA Cloning: A Practical Approach,Volumes I and II, 1985 (D. N. Glover ed.); Oligonucleotide Synthesis,1984, (M. L. Gait ed); Transcription and Translation, 1984 (Hames andHiggins eds.); Animal Cell Culture, 1986 (R. I. Freshney ed.);Immobilized Cells and Enzymes, 1986 (IRL Press); Perbal, 1984, APractical Guide to Molecular Cloning; and Gene Transfer Vectors forMammalian Cells, 1987 (J. H. Miller and M. P. Calos eds., Cold SpringHarbor Laboratory).

Any suitable materials and/or methods known to those of skill can beutilized in carrying out the present invention: however preferredmaterials and/or methods are described. Materials, reagents and the liketo which reference is made in the following description and examples areobtainable from commercial sources, unless otherwise noted.

S. pneumoniae Genomic Sequence

This invention provides nucleotide sequences of the genome of S.pneumoniae which thus comprises a DNA sequence library of S. pneumoniaegenomic DNA. The detailed description that follows provides nucleotidesequences of S. pneumoniae, and also describes how the sequences wereobtained and how ORFs and protein-coding sequences were identified. Alsodescribed are methods of using the disclosed S. pneumoniae sequences inmethods including diagnostic and therapeutic applications. Furthermore,the library can be used as a database for identification and comparisonof medically important sequences in this and other strains of S.pneumoniae.

To determine the genomic sequence of S. pneumoniae, DNA was isolatedfrom strain 14453 of S. pneumoniae and mechanically sheared bynebulization to a median size of 2 kb. Following size fractionation bygel electrophoresis, the fragments were blunt-ended, ligated to adapteroligonucleotides, and cloned into each of 20 different pMPX vectors(Rice et al., abstracts of Meeting of Genome Mapping and Sequencing,Cold Spring Harbor, N.Y., 5/11-5/15, 1994, p. 225) and the PUC19 vectorto construct a series of “shotgun” subclone libraries.

DNA sequencing was achieved using two sequencing methods. The firstmethod used multiplex sequencing procedures essentially as disclosed inChurch et al., 1988, Science 240:185; U.S. Pat. Nos. 4,942,124 and5,149,625). DNA was extracted from pooled cultures and subjected tochemical or enzymatic sequencing. Sequencing reactions were resolved byelectrophoresis, and the products were transferred and covalently boundto nylon membranes. Finally, the membranes were sequentially hybridizedwith a series of labelled oligonucleotides complimentary to “tag”sequences present in the different shotgun cloning vectors. In thismanner, a large number of sequences could be obtained from a single setof sequencing reactions. The remainder of the sequencing was performedon ABI377 automated DNA sequencers. The cloning and sequencingprocedures are described in more detail in the Exemplification.

Individual sequence reads were assembled using PHRAP (P. Green,Abstracts of DOE Human Genome Program Contractor-Grantee Workshop V,January 1996, p. 157). The average contig length was about 3-4 kb.

A variety of approaches are used to order the contigs so as to obtain acontinuous sequence representing the entire S. pneumoniae genome.Synthetic oligonucleotides are designed that are complementary tosequences at the end of each contig. These oligonucleotides may behybridized to libraries of S. pneumoniae genomic DNA in, for example,lambda phage vectors or plasmid vectors to identify clones that containsequences corresponding to the junctional regions between individualcontigs. Such clones are then used to isolate template DNA and the sameoligonucleotides are used as primers in polymerase chain reaction (PCR)to amplify junctional fragments, the nucleotide sequence of which isthen determined.

The S. pneumoniae sequences were analyzed for the presence of openreading frames (ORFs) comprising at least 180 nucleotides. As a resultof the initial analysis of ORFs based on stop-to-stop codon reads, itshould be understood that these ORFs may not correspond to the ORF of anaturally-occurring S. pneumoniae polypeptide. These ORFs may containstart codons which indicate the initiation of protein synthesis of anaturally-occurring S. pneumoniae polypeptide. Such start codons withinthe ORFs provided herein can be identified by those of ordinary skill inthe relevant art, and the resulting ORF and the encoded S. pneumoniaepolypeptide is within the scope of this invention. For example, withinthe ORFs a codon such as AUG or GUG (encoding methionine or valine)which is part of the initiation signal for protein synthesis can beidentified and the portion of an ORF to corresponding to anaturally-occurring S. pneumoniae polypeptide can be recognized.

The second analysis of the ORFs included identifying the start codonsand the predicted coding regions. These ORFs provided in this inventionwere defined by one or more of the following methods: evaluating thecoding potential of such sequences with the program GENEMARK™(Borodovsky and McIninch, 1993, Comp. 17:123), distinguishing the codingfrom noncoding regions using the program Glimmer (Fraser et al, Nature,1997), determining codon usage (Staden et al., Nucleic Acid Research 10:141), and each predicted ORF amino acid sequence was compared with allprotein sequences found in current GENBANK, SWISS-PROT, and PIRdatabases using the BLAST algorithm. BLAST identifies local alignmentsoccurring by chance between the ORF sequence and the sequence in thedatabank (Altschal et al., 1990, L Mol. Biol. 215:403-410). HomologousORFs (probabilities less than 10⁻⁵ by chance) and ORF's that areprobably non-homologous (probabilities greater than 10⁻⁵ by chance) buthave good codon usage were identified. Both homologous, sequences andnon-homologous sequences with good codon usage are likely to encodeproteins and are encompassed by the invention.

S. pneumoniae Nucleic Acids

The nucleic acids of this invention may be obtained directly from theDNA of the above referenced S. pneumoniae strain by using the polymerasechain reaction (PCR). See “PCR, A Practical Approach” (McPherson,Quirke, and Taylor, eds., IRL Press, Oxford, UK, 1991) for details aboutthe PCR. High fidelity PCR can be used to ensure a faithful DNA copyprior to expression. In addition, the authenticity of amplified productscan be verified by conventional sequencing methods. Clones carrying thedesired sequences described in this invention may also be obtained byscreening the libraries by means of the PCR or by hybridization ofsynthetic oligonucleotide probes to filter lifts of the library coloniesor plaques as known in the art (see, e.g., Sambrook et al., MolecularCloning, A Laboratory Manual 2nd edition, 1989, Cold Spring HarborPress, NY).

It is also possible to obtain nucleic acids encoding S. pneumoniaepolypeptides from a cDNA library in accordance with protocols hereindescribed. A cDNA encoding an S. pneumoniae polypeptide can be obtainedby isolating total mRNA from an appropriate strain. Double strandedcDNAs can then be prepared from the total mRNA. Subsequently, the cDNAscan be inserted into a suitable plasmid or viral (e.g., bacteriophage)vector using any one of a number of known techniques. Genes encoding S.pneumoniae polypeptides can also be cloned using established polymerasechain reaction techniques in accordance with the nucleotide sequenceinformation provided by the invention. The nucleic acids of theinvention can be DNA or RNA. Preferred nucleic acids of the inventionare contained in the Sequence Listing.

The nucleic acids of the invention can also be chemically synthesizedusing standard techniques. Various methods of chemically synthesizingpolydeoxynucleotides are known, including solid-phase synthesis which,like peptide synthesis, has been fully automated in commerciallyavailable DNA synthesizers (See e.g., Itakura et al. U.S. Pat. No.4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; and Itakura U.S.Pat. Nos. 4,401,796 and 4,373,071, incorporated by reference herein).

Nucleic acids isolated or synthesized in accordance with features of thepresent invention are useful, by way of example, without limitation, asprobes, primers, capture ligands, antisense genes and for developingexpression systems for the synthesis of proteins and peptidescorresponding to such sequences. As probes, primers, capture ligands andantisense agents, the nucleic acid normally consists of all or part(approximately twenty or more nucleotides for specificity as well as theability to form stable hybridization products) of the nucleic acids ofthe invention contained in the Sequence Listing. These uses aredescribed in further detail below.

Probes

A nucleic acid isolated or synthesized in accordance with the sequenceof the invention contained in the Sequence Listing can be used as aprobe to specifically detect S. pneumoniae. With the sequenceinformation set forth in the present application, sequences of twenty ormore nucleotides are identified which provide the desired inclusivityand exclusivity with respect to S. pneumoniae, and extraneous nucleicacids likely to be encountered during hybridization conditions. Morepreferably, the sequence will comprise at least twenty to thirtynucleotides to convey stability to the hybridization product formedbetween the probe and the intended target molecules.

Sequences larger than 1000 nucleotides in length are difficult tosynthesize but can be generated by recombinant DNA techniques.Individuals skilled in the art will readily recognize that the nucleicacids, for use as probes, can be provided with a label to facilitatedetection of a hybridization product.

Nucleic acid isolated and synthesized in accordance with the sequence ofthe invention contained in the Sequence Listing can also be useful asprobes to detect homologous regions (especially homologous genes) ofother Streptococcus species using appropriate stringency hybridizationconditions as described herein.

Capture Ligand

For use as a capture ligand, the nucleic acid selected in the mannerdescribed above with respect to probes, can be readily associated with asupport. The manner in which nucleic acid is associated with supports iswell known. Nucleic acid having twenty or more nucleotides in a sequenceof the invention contained in the Sequence Listing have utility toseparate S. pneumoniae nucleic acid from the nucleic acid of each otherand other organisms. Nucleic acid having twenty or more nucleotides in asequence of the invention contained in the Sequence Listing can alsohave utility to separate other Streptococcus species from each other andfrom other organisms. Preferably, the sequence will comprise at leasttwenty nucleotides to convey stability to the hybridization productformed between the probe and the intended target molecules. Sequenceslarger than 1000 nucleotides in length are difficult to synthesize butcan be generated by recombinant DNA techniques.

Primers

Nucleic acid isolated or synthesized in accordance with the sequencesdescribed herein have utility as primers for the amplification of S.pneumoniae nucleic acid. These nucleic acids may also have utility asprimers for the amplification of nucleic acids in other Streptococcusspecies. With respect to polymerase chain reaction (PCR) techniques,nucleic acid sequences of ≧10-15 nucleotides of the invention containedin the Sequence Listing have utility in conjunction with suitableenzymes and reagents to create copies of S. pneumoniae nucleic acid.More preferably, the sequence will comprise twenty or more nucleotidesto convey stability to the hybridization product formed between theprimer and the intended target molecules. Binding conditions of primersgreater than 100 nucleotides are more difficult to control to obtainspecificity. High fidelity PCR can be used to ensure a faithful DNA copyprior to expression. In addition, amplified products can be checked byconventional sequencing methods.

The copies can be used in diagnostic assays to detect specificsequences, including genes from S. pneumoniae and/or other Streptococcusspecies. The copies can also be incorporated into cloning and expressionvectors to generate polypeptides corresponding to the nucleic acidsynthesized by PCR, as is described in greater detail herein.

Antisense

Nucleic acid or nucleic acid-hybridizing derivatives isolated orsynthesized in accordance with the sequences described herein haveutility as antisense agents to prevent the expression of S. pneumoniaegenes. These sequences also have utility as antisense agents to preventexpression of genes of other Streptococcus species.

In one embodiment, nucleic acid or derivatives corresponding to S.pneumoniae nucleic acids is loaded into a suitable carrier such as aliposome or bacteriophage for introduction into bacterial cells. Forexample, a nucleic acid having twenty or more nucleotides is capable ofbinding to bacteria nucleic acid or bacteria messenger RNA. Preferably,the antisense nucleic acid is comprised of 20 or more nucleotides toprovide necessary stability of a hybridization product of non-naturallyoccurring nucleic acid and bacterial nucleic acid and/or bacterialmessenger RNA. Nucleic acid having a sequence greater than 1000nucleotides in length is difficult to synthesize but can be generated byrecombinant DNA techniques. Methods for loading antisense nucleic acidin liposomes is known in the art as exemplified by U.S. Pat. No.4,241,046 issued Dec. 23, 1980 to Papahadjopoulos et al.

The present invention encompasses isolated polypeptides and nucleicacids derived from S. pneumoniae that are useful as reagents fordiagnosis of bacterial infection, components of effective antibacterialvaccines, and/or as targets for antibacterial drugs, including anti-S.pneumoniae drugs.

Expression of S. pneumoniae Nucleic Acids

Table 2 provides a list of open reading frames (ORFs) in both strands.An ORF is a region of nucleic acid which encodes a polypeptide. Thisregion normally represents a complete coding sequence or a totalsequence and was determined from an initial analysis of stop to stopcodons followed by the prediction of start codons. The first columnlists the ORF designation. The second and third columns list the SEQ IDnumbers for the nucleic acid and amino acid sequences corresponding toeach ORF, respectively. The fourth and fifth columns list the length ofthe nucleic acid ORF and the length of the amino acid ORF, respectively.Most of the nucleotide sequences corresponding to each ORF begin at thefirst nucleotide of the start codon and end at the nucleotideimmediately preceding the next downstream stop codon in the same readingframe. It will be recognized by one skilled in the art that the naturaltranslation initiation sites will correspond to ATG, GTG, or TTG codonslocated within the ORFs. The natural initiation sites depend not only onthe sequence of a start codon but also on the context of the DNAsequence adjacent to the start codon. Usually, a recognizable ribosomebinding site is found within 20 nucleotides upstream from the initiationcodon. In some cases where genes are translationally coupled andcoordinately expressed together in “operons”, ribosome binding sites arenot present, but the initiation codon of a downstream gene may occurvery close to, or overlap, the stop codon of the an upstream gene in thesame operon. The correct start codons can be generally identifiedrapidly and efficiently because only a few codons need be tested. It isrecognized that the translational machinery in bacteria initiates mostpolypeptide chains with the amino acid methionine. In some cases,polypeptides are post-translationally modified, resulting in anN-terminal amino acid other than methionine in vivo. The sixth andseventh columns provide metrics for assessing the likelihood of thehomology match (determined by the BLASTP2 algorithm), as is known in theart, to the genes indicated in the description field. Specifically, thesixth column represents the “Score” for the match (a higher score is abetter match), and the seventh column represents the “P-value” for thematch (the probability that such a match could have occurred by chance;the lower the value, the more likely the match is valid). If a BLASTP2score of less than 46 was obtained, no value is reported in the tablethe “P-value”. The description field provides, where available, theaccession number (AC) or the Swissprot accession number (SP), the locusname (LN), Superfamily Classification (CL), the Organism (OR), Source ofvariant (SR), E.C. number (EC), the gene name (GN), the product name(PN), the Function Description (FN), the Map Position (MP), Left End(LE), Right End (RE), Coding Direction (DI), the Database from which thesequence originates (DB), and the description (DE) or notes (NT) foreach ORF. This information allows one of ordinary skill in the art todetermine a potential use and function for each identified codingsequence and, as a result, allows the use of the polypeptides of thepresent invention for commercial and industrial purposes.

Using the information provided in SEQ ID NO: 1-SEQ ID NO: 2661 and inTable 2 together with routine cloning and sequencing methods, one ofordinary skill in the art will be able to clone and sequence all thenucleic acid fragments of interest including open reading frames (ORFs)encoding a large variety proteins of S. pneumoniae.

Nucleic acid isolated or synthesized in accordance with the sequencesdescribed herein have utility to generate polypeptides. The nucleic acidof the invention exemplified in SEQ ID NO: 1-SEQ ID NO: 2661 and inTable 2 or fragments of said nucleic acid encoding active portions of S.pneumoniae polypeptides can be cloned into suitable vectors or used toisolate nucleic acid. The isolated nucleic acid is combined withsuitable DNA linkers and cloned into a suitable vector.

The function of a specific gene or operon can be ascertained byexpression in a bacterial strain under conditions where the activity ofthe gene product(s) specified by the gene or operon in question can bespecifically measured. Alternatively, a gene product may be produced inlarge quantities in an expressing strain for use as an antigen, anindustrial reagent, for structural studies, etc. This expression can beaccomplished in a mutant strain which lacks the activity of the gene tobe tested, or in a strain that does not produce the same geneproduct(s). This includes, but is not limited to, Eucaryotic speciessuch as the yeast Saccharomyces cerevisiae, Methanobacterium strains orother Archaea, and Eubacteria such as E. coli, B. subtilis, S. aureus,S. pneumonia or Pseudomonas putida. In some cases the expression hostwill utilize the natural S. pneumoniae promoter whereas in others, itwill be necessary to drive the gene with a promoter sequence derivedfrom the expressing organism (e.g., an E. coli beta-galactosidasepromoter for expression in E. coli).

To express a gene product using the natural S. pneumoniae promoter, aprocedure such as the following can be used. A restriction fragmentcontaining the gene of interest, together with its associated naturalpromoter element and regulatory sequences (identified using the DNAsequence data) is cloned into an appropriate recombinant plasmidcontaining an origin of replication that functions in the host organismand an appropriate selectable marker. This can be accomplished by anumber of procedures known to those skilled in the art. It is mostpreferably done by cutting the plasmid and the fragment to be clonedwith the same restriction enzyme to produce compatible ends that can beligated to join the two pieces together. The recombinant plasmid isintroduced into the host organism by, for example, electroporation andcells containing the recombinant plasmid are identified by selection forthe marker on the plasmid. Expression of the desired gene product isdetected using an assay specific for that gene product.

In the case of a gene that requires a different promoter, the body ofthe gene (coding sequence) is specifically excised and cloned into anappropriate expression plasmid. This subcloning can be done by severalmethods, but is most easily accomplished by PCR amplification of aspecific fragment and ligation into an expression plasmid after treatingthe PCR product with a restriction enzyme or exonuclease to createsuitable ends for cloning.

A suitable host cell for expression of a gene can be any procaryotic oreucaryotic cell. For example, an S. pneumoniae polypeptide can beexpressed in bacterial cells such as E. coli or B. subtilis, insectcells (baculovirus), yeast, or mammalian cells such as Chinese hamsterovary cell (CHO). Other suitable host cells are known to those skilledin the art.

Expression in eucaryotic cells such as mammalian, yeast, or insect cellscan lead to partial or complete glycosylation and/or formation ofrelevant inter- or intra-chain disulfide bonds of a recombinant peptideproduct. Examples of vectors for expression in yeast S. cerivisaeinclude pYepSec1 (Baldari. et al., (1987) Embo J. 6:229-234), pMFa(Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY 88 (Schultz etal., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, SanDiego, Calif.). Baculovirus vectors available for expression of proteinsin cultured insect cells (SF 9 cells) include the pAc series (Smith etal., (1983) Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow,V. A., and Summers, M. D., (1989) Virology 170:31-39). Generally, COScells (Gluzman, Y., (1981) Cell 23:175-182) are used in conjunction withsuch vectors as pCDM 8 (Aruffo, A. and Seed, B., (1987) Proc. Natl.Acad. Sci. USA 84:8573-8577) for transient amplification/expression inmammalian cells, while CHO (dhfr⁻ Chinese Hamster Ovary) cells are usedwith vectors such as pMT2PC (Kaufman et al. (1987), EMBO J. 6:187-195)for stable amplification/expression in mammalian cells. Vector DNA canbe introduced into mammalian cells via conventional techniques such ascalcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, or electroporation. Suitable methodsfor transforming host cells can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratorypress (1989)), and other laboratory textbooks.

Expression in procaryotes is most often carried out in E. coli witheither fusion or non-fusion inducible expression vectors. Fusion vectorsusually add a number of NH₂ terminal amino acids to the expressed targetgene. These NH₂ terminal amino acids often are referred to as a reportergroup or an affinity purification group. Such reporter groups usuallyserve two purposes: 1) to increase the solubility of the targetrecombinant protein; and 2) to aid in the purification of the targetrecombinant protein by acting as a ligand in affinity purification.Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the reporter group and the targetrecombinant protein to enable separation of the target recombinantprotein from the reporter group subsequent to purification of the fusionprotein. Such enzymes, and their cognate recognition sequences, includeFactor Xa, thrombin and enterokinase. Typical fusion expression vectorsinclude pGEX (Amrad Corp., Melbourne, Australia), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase, maltose E binding protein, or protein A,respectively, to the target recombinant protein. A preferred reportergroup is poly(His), which may be fused to the amino or carboxy terminusof the protein and which renders the recombinant fusion protein easilypurifiable by metal chelate chromatography.

Inducible non-fusion expression vectors include pTrc (Amann et al.,(1988) Gene 69:301-315) and pET11d (Studier et al., Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990) 60-89). While target gene expression relies on host RNApolymerase transcription from the hybrid trp-lac fusion promoter inpTrc, expression of target genes inserted into pET11d relies ontranscription from the T7 gn10-lac 0 fusion promoter mediated bycoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21 (DE3) or HMS174 (DE3) from a resident λprophage harboring a T7 gn1 under the transcriptional control of thelacUV 5 promoter.

For example, a host cell transfected with a nucleic acid vectordirecting expression of a nucleotide sequence encoding an S. pneumoniaepolypeptide can be cultured under appropriate conditions to allowexpression of the polypeptide to occur. The polypeptide may be secretedand isolated from a mixture of cells and medium containing the peptide.Alternatively, the polypeptide may be retained cytoplasmically and thecells harvested, lysed and the protein isolated. A cell culture includeshost cells, media and other byproducts. Suitable media for cell cultureare well known in the art. Polypeptides of the invention can be isolatedfrom cell culture medium, host cells, or both using techniques known inthe art for purifying proteins including ion-exchange chromatography,gel filtration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for suchpolypeptides. Additionally, in many situations, polypeptides can beproduced by chemical cleavage of a native protein (e.g., trypticdigestion) and the cleavage products can then be purified by standardtechniques.

In the case of membrane bound proteins, these can be isolated from ahost cell by contacting a membrane-associated protein fraction with adetergent forming a solubilized complex, where the membrane-associatedprotein is no longer entirely embedded in the membrane fraction and issolubilized at least to an extent which allows it to bechromatographically isolated from the membrane fraction. Severaldifferent criteria are used for choosing a detergent suitable forsolubilizing these complexes. For example, one property considered isthe ability of the detergent to solubilize the S. pneumoniae proteinwithin the membrane fraction at minimal denaturation of themembrane-associated protein allowing for the activity or functionalityof the membrane-associated protein to return upon reconstitution of theprotein. Another property considered when selecting the detergent is thecritical micelle concentration (CMC) of the detergent in that thedetergent of choice preferably has a high CMC value allowing for ease ofremoval after reconstitution. A third property considered when selectinga detergent is the hydrophobicity of the detergent. Typically,membrane-associated proteins are very hydrophobic and thereforedetergents which are also hydrophobic, e.g., the triton series, would beuseful for solubilizing the hydrophobic proteins. Another propertyimportant to a detergent can be the capability of the detergent toremove the S. pneumoniae protein with minimal protein-proteininteraction facilitating further purification. A fifth property of thedetergent which should be considered is the charge of the detergent. Forexample, if it is desired to use ion exchange resins in the purificationprocess then preferably detergent should be an uncharged detergent.Chromatographic techniques which can be used in the final purificationstep are known in the art and include hydrophobic interaction, lectinaffinity, ion exchange, dye affinity and immunoaffinity.

One strategy to maximize recombinant S. pneumoniae peptide expression inE. coli is to express the protein in a host bacteria with an impairedcapacity to proteolytically cleave the recombinant protein (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Another strategy would be toalter the nucleic acid encoding an S. pneumoniae peptide to be insertedinto an expression vector so that the individual codons for each aminoacid would be those preferentially utilized in highly expressed E. coliproteins (Wada et al., (1992) Nuc. Acids Res. 20:2111-2118). Suchalteration of nucleic acids of the invention can be carried out bystandard DNA synthesis techniques.

The nucleic acids of the invention can also be chemically synthesizedusing standard techniques. Various methods of chemically synthesizingpolydeoxynucleotides are known, including solid-phase synthesis which,like peptide synthesis, has been fully automated in commerciallyavailable DNA synthesizers (See, e.g., Itakura et al. U.S. Pat. No.4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; and Itakura U.S.Pat. Nos. 4,401,796 and 4,373,071, incorporated by reference herein).

The present invention provides a library of S. pneumoniae-derivednucleic acid sequences. The libraries provide probes, primers, andmarkers which can be used as markers in epidemiological studies. Thepresent invention also provides a library of S. pneumoniae-derivednucleic acid sequences which comprise or encode targets for therapeuticdrugs.

Nucleic acids comprising any of the sequences disclosed herein orsub-sequences thereof can be prepared by standard methods using thenucleic acid sequence information provided in SEQ ID NO: 1-SEQ ID NO:2661. For example, DNA can be chemically synthesized using, e.g., thephosphoramidite solid support method of Matteucci et al., 1981, J. Am.Chem. Soc. 103:3185, the method of Yoo et al., 1989, J. Biol. Chem.764:17078, or other well known methods. This can be done by sequentiallylinking a series of oligonucleotide cassettes comprising pairs ofsynthetic oligonucleotides, as described below.

Of course, due to the degeneracy of the genetic code, many differentnucleotide sequences can encode polypeptides having the amino acidsequences defined by SEQ ID NO: 2662-SEQ ID NO: 5322 or sub-sequencesthereof. The codons can be selected for optimal expression inprokaryotic or eukaryotic systems. Such degenerate variants are alsoencompassed by this invention.

Insertion of nucleic acids (typically DNAs) encoding the polypeptides ofthe invention into a vector is easily accomplished when the termini ofboth the DNAs and the vector comprise compatible restriction sites. Ifthis cannot be done, it may be necessary to modify the termini of theDNAs and/or vector by digesting back single-stranded DNA overhangsgenerated by restriction endonuclease cleavage to produce blunt ends, orto achieve the same result by filling in the single-stranded terminiwith an appropriate DNA polymerase.

Alternatively, any site desired may be produced, e.g., by ligatingnucleotide sequences (linkers) onto the termini. Such linkers maycomprise specific oligonucleotide sequences that define desiredrestriction sites. Restriction sites can also be generated by the use ofthe polymerase chain reaction (PCR). See, e.g., Saiki et al., 1988,Science 239:48. The cleaved vector and the DNA fragments may also bemodified if required by homopolymeric tailing.

In certain embodiments, the invention encompasses isolated nucleic acidfragments comprising all or part of the individual nucleic acidsequences disclosed herein. The fragments are at least about 8nucleotides in length, preferably at least about 12 nucleotides inlength, and most preferably at least about 15-20 nucleotides in length.

The nucleic acids may be isolated directly from cells. Alternatively,the polymerase chain reaction (PCR) method can be used to produce thenucleic acids of the invention, using either chemically synthesizedstrands or genomic material as templates. Primers used for PCR can besynthesized using the sequence information provided herein and canfurther be designed to introduce appropriate new restriction sites, ifdesirable, to facilitate incorporation into a given vector forrecombinant expression.

The nucleic acids of the present invention may be flanked by natural S.pneumoniae regulatory sequences, or may be associated with heterologoussequences, including promoters, enhancers, response elements, signalsequences, polyadenylation sequences, introns, 5′- and 3′-noncodingregions, and the like. The nucleic acids may also be modified by manymeans known in the art. Non-limiting examples of such modificationsinclude methylation, “caps”, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoramidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.). Nucleic acids may contain one or moreadditional covalently linked moieties, such as, for example, proteins(e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine,etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g.,metals, radioactive metals, iron, oxidative metals, etc.), andalkylators. PNAs are also included. The nucleic acid may be derivatizedby formation of a methyl or ethyl phosphotriester or an alkylphosphoramidate linkage. Furthermore, the nucleic acid sequences of thepresent invention may also be modified with a label capable of providinga detectable signal, either directly or indirectly. Exemplary labelsinclude radioisotopes, fluorescent molecules, biotin, and the like.

The invention also provides nucleic acid vectors comprising thedisclosed S. pneumoniae-derived sequences or derivatives or fragmentsthereof. A large number of vectors, including plasmid and fungalvectors, have been described for replication and/or expression in avariety of eukaryotic and prokaryotic hosts, and may be used for genetherapy as well as for simple cloning or protein expression.

The encoded S. pneumoniae polypeptides may be expressed by using manyknown vectors, such as pUC plasmids, pET plasmids (Novagen, Inc.,Madison, Wis.), or pRSET or pREP (Invitrogen, San Diego, Calif.), andmany appropriate host cells, using methods disclosed or cited herein orotherwise known to those skilled in the relevant art. The particularchoice of vector/host is not critical to the practice of the invention.

Recombinant cloning vectors will often include one or more replicationsystems for cloning or expression, one or more markers for selection inthe host, e.g. antibiotic resistance, and one or more expressioncassettes. The inserted S. pneumoniae coding sequences may besynthesized by standard methods, isolated from natural sources, orprepared as hybrids, etc. Ligation of the S. pneumoniae coding sequencesto transcriptional regulatory elements and/or to other amino acid codingsequences may be achieved by known methods. Suitable host cells may betransformed/transfected/infected as appropriate by any suitable methodincluding electroporation, CaCl₂ mediated DNA uptake, fungal infection,microinjection, microprojectile, or other established methods.

Appropriate host cells include bacteria, archebacteria, fungi,especially yeast, and plant and animal cells, especially mammaliancells. Of particular interest are S. pneumoniae, E. coli, B. Subtilis,Saccharomyces cerevisiae, Saccharomyces carisbergensis,Schizosaccharomyces pombi, SF9 cells, C129 cells, 293 cells, Neurospora,and CHO cells, COS cells, HeLa cells, and immortalized mammalian myeloidand lymphoid cell lines. Preferred replication systems include M13,ColE1, SV40, baculovirus, lambda, adenovirus, and the like. A largenumber of transcription initiation and termination regulatory regionshave been isolated and shown to be effective in the transcription andtranslation of heterologous proteins in the various hosts. Examples ofthese regions, methods of isolation, manner of manipulation, etc. areknown in the art. Under appropriate expression conditions, host cellscan be used as a source of recombinantly produced S. pneumoniae-derivedpeptides and polypeptides.

Advantageously, vectors may also include a transcription regulatoryelement (i.e., a promoter) operably linked to the S. pneumoniae portion.The promoter may optionally contain operator portions and/or ribosomebinding sites. Non-limiting examples of bacterial promoters compatiblewith E. coli include: b-lactamase (penicillinase) promoter; lactosepromoter; tryptophan (trp) promoter; araBAD (arabinose) operon promoter;lambda-derived P₁ promoter and N gene ribosome binding site; and thehybrid tac promoter derived from sequences of the trp and lac UV5promoters. Non-limiting examples of yeast promoters include3-phosphoglycerate kinase promoter, glyceraldehyde-3-phosphatedehydrogenase (GAPDH) promoter, galactokinase (GAL1) promoter,galactoepimerase promoter, and alcohol dehydrogenase (ADH) promoter.Suitable promoters for mammalian cells include without limitation viralpromoters such as that from Simian Virus 40 (SV40), Rous sarcoma virus(RSV), adenovirus (ADV), and bovine papilloma virus (BPV). Mammaliancells may also require terminator sequences, polyA addition sequencesand enhancer sequences to increase expression. Sequences which causeamplification of the gene may also be desirable. Furthermore, sequencesthat facilitate secretion of the recombinant product from cells,including, but not limited to, bacteria, yeast, and animal cells, suchas secretory signal sequences and/or prohormone pro region sequences,may also be included. These sequences are well described in the art.

Nucleic acids encoding wild-type or variant S. pneumoniae-derivedpolypeptides may also be introduced into cells by recombination events.For example, such a sequence can be introduced into a cell, and therebyeffect homologous recombination at the site of an endogenous gene or asequence with substantial identity to the gene. Otherrecombination-based methods such as nonhomologous recombinations ordeletion of endogenous genes by homologous recombination may also beused.

The nucleic acids of the present invention find use as templates for therecombinant production of S. pneumoniae-derived peptides orpolypeptides.

Identification and Use of S. pneumoniae Nucleic Acid Sequences

The disclosed S. pneumoniae polypeptide and nucleic acid sequences, orother sequences that are contained within ORFs, including completeprotein-coding sequences, of which any of the disclosed S.pneumoniae-specific sequences forms a part, are useful as targetcomponents for diagnosis and/or treatment of S. pneumoniae-causedinfection

It will be understood that the sequence of an entire protein-codingsequence of which each disclosed nucleic acid sequence forms a part canbe isolated and identified based on each disclosed sequence. This can beachieved, for example, by using an isolated nucleic acid encoding thedisclosed sequence, or fragments thereof, to prime a sequencing reactionwith genomic S. pneumoniae DNA as template; this is followed bysequencing the amplified product. The isolated nucleic acid encoding thedisclosed sequence, or fragments thereof, can also be hybridized to S.pneumoniae genomic libraries to identify clones containing additionalcomplete segments of the protein-coding sequence of which the shortersequence forms a part. Then, the entire protein-coding sequence, orfragments thereof, or nucleic acids encoding all or part of thesequence, or sequence-conservative or function-conservative variantsthereof, may be employed in practicing the present invention.

Preferred sequences are those that are useful in diagnostic and/ortherapeutic applications. Diagnostic applications include withoutlimitation nucleic-acid-based and antibody-based methods for detectingbacterial infection. Therapeutic applications include without limitationvaccines, passive immunotherapy, and drug treatments directed againstgene products that are both unique to bacteria and essential for growthand/or replication of bacteria.

Identification of Nucleic Acids Encoding Vaccine Components and Targetsfor Agents Effective Against S. pneumoniae

The disclosed S. pneumoniae genome sequence includes segments thatdirect the synthesis of ribonucleic acids and polypeptides, as well asorigins of replication, promoters, other types of regulatory sequences,and intergenic nucleic acids. The invention encompasses nucleic acidsencoding immunogenic components of vaccines and targets for agentseffective against S. pneumoniae. Identification of said immunogeniccomponents involved in the determination of the function of thedisclosed sequences, which can be achieved using a variety ofapproaches. Non-limiting examples of these approaches are describedbriefly below.

Homology to Known Sequences:

Computer-assisted comparison of the disclosed S. pneumoniae sequenceswith previously reported sequences present in publicly availabledatabases is useful for identifying functional S. pneumoniae nucleicacid and polypeptide sequences. It will be understood thatprotein-coding sequences, for example, may be compared as a whole, andthat a high degree of sequence homology between two proteins (such as,for example, >80-90%) at the amino acid level indicates that the twoproteins also possess some degree of functional homology, such as, forexample, among enzymes involved in metabolism, DNA synthesis, or cellwall synthesis, and proteins involved in transport, cell division, etc.In addition, many structural features of particular protein classes havebeen identified and correlate with specific consensus sequences, suchas, for example, binding domains for nucleotides, DNA, metal ions, andother small molecules; sites for covalent modifications such asphosphorylation, acylation, and the like; sites of protein:proteininteractions, etc. These consensus sequences may be quite short and thusmay represent only a fraction of the entire protein-coding sequence.Identification of such a feature in an S. pneumoniae sequence istherefore useful in determining the function of the encoded protein andidentifying useful targets of antibacterial drugs.

Of particular relevance to the present invention are structural featuresthat are common to secretory, transmembrane, and surface proteins,including secretion signal peptides and hydrophobic transmembranedomains. S. pneumoniae proteins identified as containing putative signalsequences and/or transmembrane domains are useful as immunogeniccomponents of vaccines.

Targets for therapeutic drugs according to the invention include, butare not limited to, polypeptides of the invention, whether unique to S.pneumoniae or not, that are essential for growth and/or viability of S.pneumoniae under at least one growth condition. Polypeptides essentialfor growth and/or viability can be determined by examining the effect ofdeleting and/or disrupting the genes, i.e., by so-called gene“knockout”. Alternatively, genetic footprinting can be used (Smith etal., 1995, Proc. Natl. Acad. Sci. USA 92:5479-6433; PublishedInternational Application WO 94/26933; U.S. Pat. No. 5,612,180). Stillother methods for assessing essentiality includes the ability to isolateconditional lethal mutations in the specific gene (e.g., temperaturesensitive mutations). Other useful targets for therapeutic drugs, whichinclude polypeptides that are not essential for growth or viability perse but lead to loss of viability of the cell, can be used to targettherapeutic agents to cells.

Strain-Specific Sequences:

Because of the evolutionary relationship between different S. pneumoniaestrains, it is believed that the presently disclosed S. pneumoniaesequences are useful for identifying, and/or discriminating between,previously known and new S. pneumoniae strains. It is believed thatother S. pneumoniae strains will exhibit at least 70% sequence homologywith the presently disclosed sequence. Systematic and routine analysesof DNA sequences derived from samples containing S. pneumoniae strains,and comparison with the present sequence allows for the identificationof sequences that can be used to discriminate between strains, as wellas those that are common to all S. pneumoniae strains. In oneembodiment, the invention provides nucleic acids, including probes, andpeptide and polypeptide sequences that discriminate between differentstrains of S. pneumoniae. Strain-specific components can also beidentified functionally by their ability to elicit or react withantibodies that selectively recognize one or more S. pneumoniae strains.

In another embodiment, the invention provides nucleic acids, includingprobes, and peptide and polypeptide sequences that are common to all S.pneumoniae strains but are not found in other bacterial species.

S. pneumoniae Polypeptides

This invention encompasses isolated S. pneumoniae polypeptides encodedby the disclosed S. pneumoniae genomic sequences, including thepolypeptides of the invention contained in the Sequence Listing.Polypeptides of the invention are preferably at least 5 amino acidresidues in length. Using the DNA sequence information provided herein,the amino acid sequences of the polypeptides encompassed by theinvention can be deduced using methods well-known in the art. It will beunderstood that the sequence of an entire nucleic acid encoding an S.pneumoniae polypeptide can be isolated and identified based on an ORFthat encodes only a fragment of the cognate protein-coding region. Thiscan be achieved, for example, by using the isolated nucleic acidencoding the ORF, or fragments thereof, to prime a polymerase chainreaction with genomic S. pneumoniae DNA as template; this is followed bysequencing the amplified product.

The polypeptides of the present invention, includingfunction-conservative variants of the disclosed ORFs, may be isolatedfrom wild-type or mutant S. pneumoniae cells, or from heterologousorganisms or cells (including, but not limited to, bacteria, fungi,insect, plant, and mammalian cells) including S. pneumoniae into which aS. pneumoniae-derived protein-coding sequence has been introduced andexpressed. Furthermore, the polypeptides may be part of recombinantfusion proteins.

S. pneumoniae polypeptides of the invention can be chemicallysynthesized using commercially automated procedures such as thosereferenced herein, including, without limitation, exclusive solid phasesynthesis, partial solid phase methods, fragment condensation orclassical solution synthesis. The polypeptides are preferably preparedby solid phase peptide synthesis as described by Merrifield, 1963, J.Am. Chem. Soc. 85:2149. The synthesis is carried out with amino acidsthat are protected at the alpha-amino terminus. Trifunctional aminoacids with labile side-chains are also protected with suitable groups toprevent undesired chemical reactions from occurring during the assemblyof the polypeptides. The alpha-amino protecting group is selectivelyremoved to allow subsequent reaction to take place at theamino-terminus. The conditions for the removal of the alpha-aminoprotecting group do not remove the side-chain protecting groups.

The alpha-amino protecting groups are those known to be useful in theart of stepwise polypeptide synthesis. Included are acyl type protectinggroups, e.g., formyl, trifluoroacetyl, acetyl, aromatic urethane typeprotecting groups, e.g., benzyloxycarbonyl (Cbz), substitutedbenzyloxycarbonyl and 9-fluorenylmethyloxycarbonyl (Fmoc), aliphaticurethane protecting groups, e.g., t-butyloxycarbonyl (Boc),isopropyloxycarbonyl, cyclohexyloxycarbonyl, and alkyl type protectinggroups, e.g., benzyl, triphenylmethyl. The preferred protecting group isBoc. The side-chain protecting groups for Tyr include tetrahydropyranyl,tert-butyl, trityl, benzyl, Cbz, 4-Br-Cbz and 2,6-dichlorobenzyl. Thepreferred side-chain protecting group for Tyr is 2,6-dichlorobenzyl. Theside-chain protecting groups for Asp include benzyl, 2,6-dichlorobenzyl,methyl, ethyl and cyclohexyl. The preferred side-chain protecting groupfor Asp is cyclohexyl. The side-chain protecting groups for Thr and Serinclude acetyl, benzoyl, trityl, tetrahydropyranyl, benzyl,2,6-dichlorobenzyl and Cbz. The preferred protecting group for Thr andSer is benzyl. The side-chain protecting groups for Arg include nitro,Tos, Cbz, adamantyloxycarbonyl and Boc. The preferred protecting groupfor Arg is Tos. The side-chain amino group of Lys may be protected withCbz, 2-Cl-Cbz, Tos or Boc. The 2-Cl-Cbz group is the preferredprotecting group for Lys.

The side-chain protecting groups selected must remain intact duringcoupling and not be removed during the deprotection of theamino-terminus protecting group or during coupling conditions. Theside-chain protecting groups must also be removable upon the completionof synthesis, using reaction conditions that will not alter the finishedpolypeptide.

Solid phase synthesis is usually carried out from the carboxy-terminusby coupling the alpha-amino protected (side-chain protected) amino acidto a suitable solid support. An ester linkage is formed when theattachment is made to a chloromethyl or hydroxymethyl resin, and theresulting polypeptide will have a free carboxyl group at the C-terminus.Alternatively, when a benzhydrylamine or p-methylbenzhydrylamine resinis used, an amide bond is formed and the resulting polypeptide will havea carboxamide group at the C-terminus. These resins are commerciallyavailable, and their preparation was described by Stewart et al., 1984,Solid Phase Peptide Synthesis (2nd Edition), Pierce Chemical Co.,Rockford, Ill.

The C-terminal amino acid, protected at the side chain if necessary andat the alpha-amino group, is coupled to the benzhydrylamine resin usingvarious activating agents including dicyclohexylcarbodiimide (DCC),N,N′-diisopropyl-carbodiimide and carbonyldiimidazole. Following theattachment to the resin support, the alpha-amino protecting group isremoved using trifluoroacetic acid (TFA) or HCl in dioxane at atemperature between 0 and 25° C. Dimethylsulfide is added to the TFAafter the introduction of methionine (Met) to suppress possibleS-alkylation. After removal of the alpha-amino protecting group, theremaining protected amino acids are coupled stepwise in the requiredorder to obtain the desired sequence.

Various activating agents can be used for the coupling reactionsincluding DCC, N,N′-diisopropyl-carbodiimide,benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexa-fluorophosphate (BOP) and DCC-hydroxybenzotriazole (HOBt). Eachprotected amino acid is used in excess (>2.0 equivalents), and thecouplings are usually carried out in N-methylpyrrolidone (NMP) or inDMF, CH₂Cl₂ or mixtures thereof. The extent of completion of thecoupling reaction is monitored at each stage, e.g., by the ninhydrinreaction as described by Kaiser et al., 1970, Anal. Biochem. 34:595. Incases where incomplete coupling is found, the coupling reaction isrepeated. The coupling reactions can be performed automatically withcommercially available instruments.

After the entire assembly of the desired polypeptide, thepolypeptide-resin is cleaved with a reagent such as liquid HF for 1-2hours at 0° C., which cleaves the polypeptide from the resin and removesall side-chain protecting groups. A scavenger such as anisole is usuallyused with the liquid HF to prevent cations formed during the cleavagefrom alkylating the amino acid residues present in the polypeptide. Thepolypeptide-resin may be deprotected with TFA/dithioethane prior tocleavage if desired.

Side-chain to side-chain cyclization on the solid support requires theuse of an orthogonal protection scheme which enables selective cleavageof the side-chain functions of acidic amino acids (e.g., Asp) and thebasic amino acids (e.g., Lys). The 9-fluorenylmethyl (Fm) protectinggroup for the side-chain of Asp and the 9-fluorenylmethyloxycarbonyl(Fmoc) protecting group for the side-chain of Lys can be used for thispurpose. In these cases, the side-chain protecting groups of theBoc-protected polypeptide-resin are selectively removed with piperidinein DMF. Cyclization is achieved on the solid support using variousactivating agents including DCC, DCC/HOBt or BOP. The HF reaction iscarried out on the cyclized polypeptide-resin as described above.

Methods for polypeptide purification are well-known in the art,including, without limitation, preparative disc-gel electrophoresis,isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ionexchange and partition chromatography, and countercurrent distribution.For some purposes, it is preferable to produce the polypeptide in arecombinant system in which the S. pneumoniae protein contains anadditional sequence tag that facilitates purification, such as, but notlimited to, a polyhistidine sequence. The polypeptide can then bepurified from a crude lysate of the host cell by chromatography on anappropriate solid-phase matrix. Alternatively, antibodies producedagainst a S. pneumoniae protein or against peptides derived therefromcan be used as purification reagents. Other purification methods arepossible.

The present invention also encompasses derivatives and homologues of S.pneumoniae-encoded polypeptides. For some purposes, nucleic acidsequences encoding the peptides may be altered by substitutions,additions, or deletions that provide for functionally equivalentmolecules, i.e., function-conservative variants. For example, one ormore amino acid residues within the sequence can be substituted byanother amino acid of similar properties, such as, for example,positively charged amino acids (arginine, lysine, and histidine);negatively charged amino acids (aspartate and glutamate); polar neutralamino acids; and non-polar amino acids. The isolated polypeptides may bemodified by, for example, phosphorylation, sulfation, acylation, orother protein modifications. They may also be modified with a labelcapable of providing a detectable signal, either directly or indirectly,including, but not limited to, radioisotopes and fluorescent compounds.

To identify S. pneumoniae-derived polypeptides for use in the presentinvention, essentially the complete genomic sequence of a virulent,methicillin-resistant isolate of Streptococcus pneumoniae isolate wasanalyzed. While, in very rare instances, a nucleic acid sequencing errormay be revealed, resolving a rare sequencing error is well within theart, and such an occurrence will not prevent one skilled in the art frompracticing the invention.

Also encompassed are any S. pneumoniae polypeptide sequences that arecontained within the open reading frames (ORFs), including completeprotein-coding sequences, of which any of SEQ ID NO: 2662-SEQ ID NO:5322 forms a part. Table 2, which is appended herewith and which formspart of the present specification, provides a putative identification ofthe particular function of a polypeptide which is encoded by each ORF.As a result, one skilled in the art can use the polypeptides of thepresent invention for commercial and industrial purposes consistent withthe type of putative identification of the polypeptide.

The present invention provides a library of S. Pneumoniae-derivedpolypeptide sequences, and a corresponding library of nucleic acidsequences encoding the polypeptides, wherein the polypeptidesthemselves, or polypeptides contained within ORFs of which they form apart, comprise sequences that are contemplated for use as components ofvaccines. Non-limiting examples of such sequences are listed by SEQ IDNO in Table 2, which is appended herewith and which forms part of thepresent specification.

The present invention also provides a library of S. pneumoniae-derivedpolypeptide sequences, and a corresponding library of nucleic acidsequences encoding the polypeptides, wherein the polypeptidesthemselves, or polypeptides contained within ORFs of which they form apart, comprise sequences lacking homology to any known prokaryotic oreukaryotic sequences. Such libraries provide probes, primers, andmarkers which can be used to diagnose S. pneumoniae infection, includinguse as markers in epidemiological studies. Non-limiting examples of suchsequences are listed by SEQ ID NO in Table 2, which is appended

The present invention also provides a library of S. pneumoniae-derivedpolypeptide sequences, and a corresponding library of nucleic acidsequences encoding the polypeptides, wherein the polypeptidesthemselves, or polypeptides contained within ORFs of which they form apart, comprise targets for therapeutic drugs.

SPECIFIC EXAMPLE Determination of Candidate Protein Antigens forAntibody and Vaccine Development

The selection of candidate protein antigens for vaccine development canbe derived from the nucleic acids encoding S. pneumoniae polypeptides.First, the ORF's can be analyzed for homology to other known exported ormembrane proteins and analyzed using the discriminant analysis describedby Klein, et al. (Klein, P., Kanehsia, M., and DeLisi, C. (1985)Biochimica et Biophysica Acta 815, 468-476) for predicting exported andmembrane proteins.

Homology searches can be performed using the BLAST algorithm containedin the Wisconsin Sequence Analysis Package (Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711) tocompare each predicted ORF amino acid sequence with all sequences foundin the current GenBank, SWISS-PROT and PIR databases. BLAST searches forlocal alignments between the ORF and the databank sequences and reportsa probability score which indicates the probability of finding thissequence by chance in the database. ORF's with significant homology(e.g. probabilities lower than 1×10⁻⁶ that the homology is only due torandom chance) to membrane or exported proteins represent proteinantigens for vaccine development. Possible functions can be provided toS. pneumoniae genes based on sequence homology to genes cloned in otherorganisms.

Discriminant analysis (Klein, et al. supra) can be used to examine theORF amino acid sequences. This algorithm uses the intrinsic informationcontained in the ORF amino acid sequence and compares it to informationderived from the properties of known membrane and exported proteins.This comparison predicts which proteins will be exported, membraneassociated or cytoplasmic. ORF amino acid sequences identified asexported or membrane associated by this algorithm are likely proteinantigens for vaccine development.

Production of Fragments and Analogs of S. pneumoniae Nucleic Acids andPolypeptides

Based on the discovery of the S. pneumoniae gene products of theinvention provided in the Sequence Listing, one skilled in the art canalter the disclosed structure (of S. pneumoniae genes), e.g., byproducing fragments or analogs, and test the newly produced structuresfor activity. Examples of techniques known to those skilled in therelevant art which allow the production and testing of fragments andanalogs are discussed below. These, or analogous methods can be used tomake and screen libraries of polypeptides, e.g., libraries of randompeptides or libraries of fragments or analogs of cellular proteins forthe ability to bind S. pneumoniae polypeptides. Such screens are usefulfor the identification of inhibitors of S. pneumoniae.

Generation of Fragments

Fragments of a protein can be produced in several ways, e.g.,recombinantly, by proteolytic digestion, or by chemical synthesis.Internal or terminal fragments of a polypeptide can be generated byremoving one or more nucleotides from one end (for a terminal fragment)or both ends (for an internal fragment) of a nucleic acid which encodesthe polypeptide. Expression of the mutagenized DNA produces polypeptidefragments. Digestion with “end-nibbling” endonucleases can thus generateDNA's which encode an array of fragments. DNA's which encode fragmentsof a protein can also be generated by random shearing, restrictiondigestion or a combination of the above-discussed methods.

Fragments can also be chemically synthesized using techniques known inthe art such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. For example, peptides of the present invention may bearbitrarily divided into fragments of desired length with no overlap ofthe fragments, or divided into overlapping fragments of a desiredlength.

Alteration of Nucleic Acids and Polypeptides: Random Methods

Amino acid sequence variants of a protein can be prepared by randommutagenesis of DNA which encodes a protein or a particular domain orregion of a protein. Useful methods include PCR mutagenesis andsaturation mutagenesis. A library of random amino acid sequence variantscan also be generated by the synthesis of a set of degenerateoligonucleotide sequences. (Methods for screening proteins in a libraryof variants are elsewhere herein).

PCR Mutagenesis

In PCR mutagenesis, reduced Taq polymerase fidelity is used to introducerandom mutations into a cloned fragment of DNA (Leung et al., 1989,Technique 1:11-15). The DNA region to be mutagenized is amplified usingthe polymerase chain reaction (PCR) under conditions that reduce thefidelity of DNA synthesis by Taq DNA polymerase, e.g., by using adGTP/dATP ratio of five and adding Mn²⁺ to the PCR reaction. The pool ofamplified DNA fragments are inserted into appropriate cloning vectors toprovide random mutant libraries.

Saturation Mutagenesis

Saturation mutagenesis allows for the rapid introduction of a largenumber of single base substitutions into cloned DNA fragments (Mayers etal., 1985, Science 229:242). This technique includes generation ofmutations, e.g., by chemical treatment or irradiation of single-strandedDNA in vitro, and synthesis of a complimentary DNA strand. The mutationfrequency can be modulated by modulating the severity of the treatment,and essentially all possible base substitutions can be obtained. Becausethis procedure does not involve a genetic selection for mutant fragmentsboth neutral substitutions, as well as those that alter function, areobtained. The distribution of point mutations is not biased towardconserved sequence elements.

Degenerate Oligonucleotides

A library of homologs can also be generated from a set of degenerateoligonucleotide sequences. Chemical synthesis of a degenerate sequencescan be carried out in an automatic DNA synthesizer, and the syntheticgenes then ligated into an appropriate expression vector. The synthesisof degenerate oligonucleotides is known in the art (see for example,Narang, S A (1983) Tetrahedron 39:3; Itakura et al. (1981) RecombinantDNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AG Walton,Amsterdam: Elsevier pp 273-289; Itakura et al. (1984) Annu. Rev.Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.(1983) Nucleic Acid Res. 11:477. Such techniques have been employed inthe directed evolution of other proteins (see, for example, Scott et al.(1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433;Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

Alteration of Nucleic Acids and Polypeptides: Methods for DirectedMutagenesis

Non-random or directed, mutagenesis techniques can be used to providespecific sequences or mutations in specific regions. These techniquescan be used to create variants which include, e.g., deletions,insertions, or substitutions, of residues of the known amino acidsequence of a protein. The sites for mutation can be modifiedindividually or in series, e.g., by (1) substituting first withconserved amino acids and then with more radical choices depending uponresults achieved, (2) deleting the target residue, or (3) insertingresidues of the same or a different class adjacent to the located site,or combinations of options 1-3.

Alanine Scanning Mutagenesis

Alanine scanning mutagenesis is a useful method for identification ofcertain residues or regions of the desired protein that are preferredlocations or domains for mutagenesis, Cunningham and Wells (Science244:1081-1085, 1989). In alanine scanning, a residue or group of targetresidues are identified (e.g., charged residues such as Arg, Asp, His,Lys, and Glu) and replaced by a neutral or negatively charged amino acid(most preferably alanine or polyalanine). Replacement of an amino acidcan affect the interaction of the amino acids with the surroundingaqueous environment in or outside the cell. Those domains demonstratingfunctional sensitivity to the substitutions are then refined byintroducing further or other variants at or for the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to optimize the performance of amutation at a given site, alanine scanning or random mutagenesis may beconducted at the target codon or region and the expressed desiredprotein subunit variants are screened for the optimal combination ofdesired activity.

Oligonucleotide-Mediated Mutagenesis

Oligonucleotide-mediated mutagenesis is a useful method for preparingsubstitution, deletion, and insertion variants of DNA, see, e.g.,Adelman et al., (DNA 2:183, 1983). Briefly, the desired DNA is alteredby hybridizing an oligonucleotide encoding a mutation to a DNA template,where the template is the single-stranded form of a plasmid orbacteriophage containing the unaltered or native DNA sequence of thedesired protein. After hybridization, a DNA polymerase is used tosynthesize an entire second complementary strand of the template thatwill thus incorporate the oligonucleotide primer, and will code for theselected alteration in the desired protein DNA. Generally,oligonucleotides of at least 25 nucleotides in length are used. Anoptimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al. (Proc.Natl. Acad. Sci. USA, 75: 5765 [1978]).

Cassette Mutagenesis

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al. (Gene, 34:315 [1985]). Thestarting material is a plasmid (or other vector) which includes theprotein subunit DNA to be mutated. The codon(s) in the protein subunitDNA to be mutated are identified. There must be a unique restrictionendonuclease site on each side of the identified mutation site(s). If nosuch restriction sites exist, they may be generated using theabove-described oligonucleotide-mediated mutagenesis method to introducethem at appropriate locations in the desired protein subunit DNA. Afterthe restriction sites have been introduced into the plasmid, the plasmidis cut at these sites to linearize it. A double-stranded oligonucleotideencoding the sequence of the DNA between the restriction sites butcontaining the desired mutation(s) is synthesized using standardprocedures. The two strands are synthesized separately and thenhybridized together using standard techniques. This double-strandedoligonucleotide is referred to as the cassette. This cassette isdesigned to have 3′ and 5′ ends that are comparable with the ends of thelinearized plasmid, such that it can be directly ligated to the plasmid.This plasmid now contains the mutated desired protein subunit DNAsequence.

Combinatorial Mutagenesis

Combinatorial mutagenesis can also be used to generate mutants (Ladneret al., WO 88/06630). In this method, the amino acid sequences for agroup of homologs or other related proteins are aligned, preferably topromote the highest homology possible. All of the amino acids whichappear at a given position of the aligned sequences can be selected tocreate a degenerate set of combinatorial sequences. The variegatedlibrary of variants is generated by combinatorial mutagenesis at thenucleic acid level, and is encoded by a variegated gene library. Forexample, a mixture of synthetic oligonucleotides can be enzymaticallyligated into gene sequences such that the degenerate set of potentialsequences are expressible as individual peptides, or alternatively, as aset of larger fusion proteins containing the set of degeneratesequences.

Other Modifications of S. pneumoniae Nucleic Acids and Polypeptides

It is possible to modify the structure of an S. pneumoniae polypeptidefor such purposes as increasing solubility, enhancing stability (e.g.,shelf life ex vivo and resistance to proteolytic degradation in vivo). Amodified S. pneumoniae protein or peptide can be produced in which theamino acid sequence has been altered, such as by amino acidsubstitution, deletion, or addition as described herein.

An S. pneumoniae peptide can also be modified by substitution ofcysteine residues preferably with alanine, serine, threonine, leucine orglutamic acid residues to minimize dimerization via disulfide linkages.In addition, amino acid side chains of fragments of the protein of theinvention can be chemically modified. Another modification iscyclization of the peptide.

In order to enhance stability and/or reactivity, an S. pneumoniaepolypeptide can be modified to incorporate one or more polymorphisms inthe amino acid sequence of the protein resulting from any naturalallelic variation. Additionally, D-amino acids, non-natural amino acids,or non-amino acid analogs can be substituted or added to produce amodified protein within the scope of this invention. Furthermore, an S.pneumoniae polypeptide can be modified using polyethylene glycol (PEG)according to the method of A. Sehon and co-workers (Wie et al., supra)to produce a protein conjugated with PEG. In addition, PEG can be addedduring chemical synthesis of the protein. Other modifications of S.pneumoniae proteins include reduction/alkylation (Tarr, Methods ofprotein Microcharacterization, J. E. Silver ed., Humana Press, CliftonN.J. 155-194 (1986)); acylation (Tarr, supra); chemical coupling to anappropriate carrier (Mishell and Shiigi, eds, Selected Methods inCellular Immunology, WH Freeman, San Francisco, Calif. (1980), U.S. Pat.No. 4,939,239; or mild formalin treatment (Marsh, (1971) Int. Arch. ofAllergy and Appl. Immunol., 41: 199-215).

To facilitate purification and potentially increase solubility of an S.pneumoniae protein or peptide, it is possible to add an amino acidfusion moiety to the peptide backbone. For example, hexa-histidine canbe added to the protein for purification by immobilized metal ionaffinity chromatography (Hochuli, E. et al., (1988) Bio/Technology, 6:1321-1325). In addition, to facilitate isolation of peptides free ofirrelevant sequences, specific endoprotease cleavage sites can beintroduced between the sequences of the fusion moiety and the peptide.

To potentially aid proper antigen processing of epitopes within an S.pneumoniae polypeptide, canonical protease sensitive sites can beengineered between regions, each comprising at least one epitope viarecombinant or synthetic methods. For example, charged amino acid pairs,such as KK or RR, can be introduced between regions within a protein orfragment during recombinant construction thereof. The resulting peptidecan be rendered sensitive to cleavage by cathepsin and/or othertrypsin-like enzymes which would generate portions of the proteincontaining one or more epitopes. In addition, such charged amino acidresidues can result in an increase in the solubility of the peptide.

Primary Methods for Screening Polypeptides and Analogs

Various techniques are known in the art for screening generated mutantgene products. Techniques for screening large gene libraries ofteninclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the genes under conditions in which detection of adesired activity, e.g., in this case, binding to S. pneumoniaepolypeptide or an interacting protein, facilitates relatively easyisolation of the vector encoding the gene whose product was detected.Each of the techniques described below is amenable to high through-putanalysis for screening large numbers of sequences created, e.g., byrandom mutagenesis techniques.

Two Hybrid Systems

Two hybrid assays such as the system described above (as with the otherscreening methods described herein), can be used to identifypolypeptides, e.g., fragments or analogs of a naturally-occurring S.pneumoniae polypeptide, e.g., of cellular proteins, or of randomlygenerated polypeptides which bind to an S. pneumoniae protein. (The S.pneumoniae domain is used as the bait protein and the library ofvariants are expressed as prey fusion proteins.) In an analogousfashion, a two hybrid assay (as with the other screening methodsdescribed herein), can be used to find polypeptides which bind a S.pneumoniae polypeptide.

Display Libraries

In one approach to screening assays, the candidate peptides aredisplayed on the surface of a cell or viral particle, and the ability ofparticular cells or viral particles to bind an appropriate receptorprotein via the displayed product is detected in a “panning assay”. Forexample, the gene library can be cloned into the gene for a surfacemembrane protein of a bacterial cell, and the resulting fusion proteindetected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991)Bio/Technology 9:1370-1371; and Goward et al. (1992) TIBS 18:136-140).In a similar fashion, a detectably labeled ligand can be used to scorefor potentially functional peptide homologs. Fluorescently labeledligands, e.g., receptors, can be used to detect homologs which retainligand-binding activity. The use of fluorescently labeled ligands,allows cells to be visually inspected and separated under a fluorescencemicroscope, or, where the morphology of the cell permits, to beseparated by a fluorescence-activated cell sorter.

A gene library can be expressed as a fusion protein on the surface of aviral particle. For instance, in the filamentous phage system, foreignpeptide sequences can be expressed on the surface of infectious phage,thereby conferring two significant benefits. First, since these phagecan be applied to affinity matrices at concentrations well over 10¹³phage per milliliter, a large number of phage can be screened at onetime. Second, since each infectious phage displays a gene product on itssurface, if a particular phage is recovered from an affinity matrix inlow yield, the phage can be amplified by another round of infection. Thegroup of almost identical E. coli filamentous phages M13, fd., and f1are most often used in phage display libraries. Either of the phage gIIIor gVIII coat proteins can be used to generate fusion proteins withoutdisrupting the ultimate packaging of the viral particle. Foreignepitopes can be expressed at the NH₂-terminal end of pIII and phagebearing such epitopes recovered from a large excess of phage lackingthis epitope (Ladner et al. PCT publication WO 90/02909; Garrard et al.,PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem.267:16007-16010; Griffiths et al. (1993) EMBO J 12:725-734; Clackson etal. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS89:4457-4461).

A common approach uses the maltose receptor of E. coli (the outermembrane protein, LamB) as a peptide fusion partner (Charbit et al.(1986) EMBO 5, 3029-3037). Oligonucleotides have been inserted intoplasmids encoding the LamB gene to produce peptides fused into one ofthe extracellular loops of the protein. These peptides are available forbinding to ligands, e.g., to antibodies, and can elicit an immuneresponse when the cells are administered to animals. Other cell surfaceproteins, e.g., OmpA (Schorr et al. (1991) Vaccines 91, pp. 387-392),PhoE (Agterberg, et al. (1990) Gene 88, 37-45), and PAL (Fuchs et al.(1991) Bio/Tech 9, 1369-1372), as well as large bacterial surfacestructures have served as vehicles for peptide display. Peptides can befused to pilin, a protein which polymerizes to form the pilus-a conduitfor interbacterial exchange of genetic information (Thiry et al. (1989)Appl. Environ. Microbiol. 55, 984-993). Because of its role ininteracting with other cells, the pilus provides a useful support forthe presentation of peptides to the extracellular environment. Anotherlarge surface structure used for peptide display is the bacterial motiveorgan, the flagellum. Fusion of peptides to the subunit proteinflagellin offers a dense array of many peptide copies on the host cells(Kuwajima et al. (1988) Bio/Tech. 6, 1080-1083). Surface proteins ofother bacterial species have also served as peptide fusion partners.Examples include the Staphylococcus protein A and the outer membrane IgAprotease of Neisseria (Hansson et al. (1992) J. Bacteriol. 174,4239-4245 and Klauser et al. (1990) EMBO J. 9, 1991-1999).

In the filamentous phage systems and the LamB system described above,the physical link between the peptide and its encoding DNA occurs by thecontainment of the DNA within a particle (cell or phage) that carriesthe peptide on its surface. Capturing the peptide captures the particleand the DNA within. An alternative scheme uses the DNA-binding proteinLacI to form a link between peptide and DNA (Cull et al. (1992) PNAS USA89:1865-1869). This system uses a plasmid containing the LacI gene withan oligonucleotide cloning site at its 3′-end. Under the controlledinduction by arabinose, a LacI-peptide fusion protein is produced. Thisfusion retains the natural ability of LacI to bind to a short DNAsequence known as LacO operator (LacO). By installing two copies of LacOon the expression plasmid, the LacI-peptide fusion binds tightly to theplasmid that encoded it. Because the plasmids in each cell contain onlya single oligonucleotide sequence and each cell expresses only a singlepeptide sequence, the peptides become specifically and stablelyassociated with the DNA sequence that directed its synthesis. The cellsof the library are gently lysed and the peptide-DNA complexes areexposed to a matrix of immobilized receptor to recover the complexescontaining active peptides. The associated plasmid DNA is thenreintroduced into cells for amplification and DNA sequencing todetermine the identity of the peptide ligands. As a demonstration of thepractical utility of the method, a large random library ofdodecapeptides was made and selected on a monoclonal antibody raisedagainst the opioid peptide dynorphin B. A cohort of peptides wasrecovered, all related by a consensus sequence corresponding to asix-residue portion of dynorphin B. (Cull et al. (1992) Proc. Natl.Acad. Sci. U.S.A. 89-1869)

This scheme, sometimes referred to as peptides-on-plasmids, differs intwo important ways from the phage display methods. First, the peptidesare attached to the C-terminus of the fusion protein, resulting in thedisplay of the library members as peptides having free carboxy termini.Both of the filamentous phage coat proteins, pIII and pVIII, areanchored to the phage through their C-termini, and the guest peptidesare placed into the outward-extending N-terminal domains. In somedesigns, the phage-displayed peptides are presented right at the aminoterminus of the fusion protein. (Cwirla, et al. (1990) Proc. Natl. Acad.Sci. U.S.A. 87, 6378-6382) A second difference is the set of biologicalbiases affecting the population of peptides actually present in thelibraries. The LacI fusion molecules are confined to the cytoplasm ofthe host cells. The phage coat fusions are exposed briefly to thecytoplasm during translation but are rapidly secreted through the innermembrane into the periplasmic compartment, remaining anchored in themembrane by their C-terminal hydrophobic domains, with the N-termini,containing the peptides, protruding into the periplasm while awaitingassembly into phage particles. The peptides in the LacI and phagelibraries may differ significantly as a result of their exposure todifferent proteolytic activities. The phage coat proteins requiretransport across the inner membrane and signal peptidase processing as aprelude to incorporation into phage. Certain peptides exert adeleterious effect on these processes and are underrepresented in thelibraries (Gallop et al. (1994) J. Med. Chem. 37(9):1233-1251). Theseparticular biases are not a factor in the LacI display system.

The number of small peptides available in recombinant random librariesis enormous. Libraries of 10⁷-10⁹ independent clones are routinelyprepared. Libraries as large as 10¹¹ recombinants have been created, butthis size approaches the practical limit for clone libraries. Thislimitation in library size occurs at the step of transforming the DNAcontaining randomized segments into the host bacterial cells. Tocircumvent this limitation, an in vitro system based on the display ofnascent peptides in polysome complexes has recently been developed. Thisdisplay library method has the potential of producing libraries 3-6orders of magnitude larger than the currently available phage/phagemidor plasmid libraries. Furthermore, the construction of the libraries,expression of the peptides, and screening, is done in an entirelycell-free format.

In one application of this method (Gallop et al. (1994) J. Med. Chem.37(9):1233-1251), a molecular DNA library encoding 10¹² decapeptides wasconstructed and the library expressed in an E. coli S30 in vitro coupledtranscription/translation system. Conditions were chosen to stall theribosomes on the mRNA, causing the accumulation of a substantialproportion of the RNA in polysomes and yielding complexes containingnascent peptides still linked to their encoding RNA. The polysomes aresufficiently robust to be affinity purified on immobilized receptors inmuch the same way as the more conventional recombinant peptide displaylibraries are screened. RNA from the bound complexes is recovered,converted to cDNA, and amplified by PCR to produce a template for thenext round of synthesis and screening. The polysome display method canbe coupled to the phage display system. Following several rounds ofscreening, cDNA from the enriched pool of polysomes was cloned into aphagemid vector. This vector serves as both a peptide expression vector,displaying peptides fused to the coat proteins, and as a DNA sequencingvector for peptide identification. By expressing the polysome-derivedpeptides on phage, one can either continue the affinity selectionprocedure in this format or assay the peptides on individual clones forbinding activity in a phage ELISA, or for binding specificity in acompletion phage ELISA (Barret, et al. (1992) Anal. Biochem 204,357-364). To identify the sequences of the active peptides one sequencesthe DNA produced by the phagemid host.

Secondary Screening of Polypeptides and Analogs

The high through-put assays described above can be followed by secondaryscreens in order to identify further biological activities which will,e.g., allow one skilled in the art to differentiate agonists fromantagonists. The type of a secondary screen used will depend on thedesired activity that needs to be tested. For example, an assay can bedeveloped in which the ability to inhibit an interaction between aprotein of interest and its respective ligand can be used to identifyantagonists from a group of peptide fragments isolated though one of theprimary screens described above.

Therefore, methods for generating fragments and analogs and testing themfor activity are known in the art. Once the core sequence of interest isidentified, it is routine for one skilled in the art to obtain analogsand fragments.

Peptide Mimetics of S. pneumoniae Polypeptides

The invention also provides for reduction of the protein binding domainsof the subject S. pneumoniae polypeptides to generate mimetics, e.g.peptide or non-peptide agents. The peptide mimetics are able to disruptbinding of a polypeptide to its counter ligand, e.g., in the case of anS. pneumoniae polypeptide binding to a naturally occurring ligand. Thecritical residues of a subject S. pneumoniae polypeptide which areinvolved in molecular recognition of a polypeptide can be determined andused to generate S. pneumoniae-derived peptidomimetics whichcompetitively or noncompetitively inhibit binding of the S. pneumoniaepolypeptide with an interacting polypeptide (see, for example, Europeanpatent applications EP-412,762A and EP-B31,080A).

For example, scanning mutagenesis can be used to map the amino acidresidues of a particular S. pneumoniae polypeptide involved in bindingan interacting polypeptide, peptidomimetic compounds (e.g. diazepine orisoquinoline derivatives) can be generated which mimic those residues inbinding to an interacting polypeptide, and which therefore can inhibitbinding of an S. pneumoniae polypeptide to an interacting polypeptideand thereby interfere with the function of S. pneumoniae polypeptide.For instance, non-hydrolyzable peptide analogs of such residues can begenerated using benzodiazepine (e.g., see Freidinger et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), substituted gama lactam rings (Garvey et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson etal. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structureand Function (Proceedings of the 9th American Peptide Symposium) PierceChemical Co. Rockland, Ill., 1985), b-turn dipeptide cores (Nagai et al.(1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc PerkinTrans 1:1231), and b-aminoalcohols (Gordon et al. (1985) Biochem BiophysRes Commun 126:419; and et al. (1986) Biochem Biophys Res Commun134:71).

Vaccine Formulations for S. pneumoniae Nucleic Acids and Polypeptides

This invention also features vaccine compositions for protection againstinfection by S. pneumoniae or for treatment of S. pneumoniae infection,a gram-negative spiral microaerophilic bacterium. In one embodiment, thevaccine compositions contain one or more immunogenic components such asa surface protein from S. pneumoniae, or portion thereof, and apharmaceutically acceptable carrier. Nucleic acids within the scope ofthe invention are exemplified by the nucleic acids of the inventioncontained in the Sequence Listing which encode S. pneumoniae surfaceproteins. Any nucleic acid encoding an immunogenic S. pneumoniaeprotein, or portion thereof, which is capable of expression in a cell,can be used in the present invention. These vaccines have therapeuticand prophylactic utilities.

One aspect of the invention provides a vaccine composition forprotection against infection by S. pneumoniae which contains at leastone immunogenic fragment of an S. pneumoniae protein and apharmaceutically acceptable carrier. Preferred fragments includepeptides of at least about 10 amino acid residues in length, preferablyabout 10-20 amino acid residues in length, and more preferably about12-16 amino acid residues in length.

Immunogenic components of the invention can be obtained, for example, byscreening polypeptides recombinantly produced from the correspondingfragment of the nucleic acid encoding the full-length S. pneumoniaeprotein. In addition, fragments can be chemically synthesized usingtechniques known in the art such as conventional Merrifield solid phasef-Moc or t-Boc chemistry.

In one embodiment, immunogenic components are identified by the abilityof the peptide to stimulate T cells. Peptides which stimulate T cells,as determined by, for example, T cell proliferation or cytokinesecretion are defined herein as comprising at least one T cell epitope.T cell epitopes are believed to be involved in initiation andperpetuation of the immune response to the protein allergen which isresponsible for the clinical symptoms of allergy. These T cell epitopesare thought to trigger early events at the level of the T helper cell bybinding to an appropriate HLA molecule on the surface of an antigenpresenting cell, thereby stimulating the T cell subpopulation with therelevant T cell receptor for the epitope. These events lead to T cellproliferation, lymphokine secretion, local inflammatory reactions,recruitment of additional immune cells to the site of antigen/T cellinteraction, and activation of the B cell cascade, leading to theproduction of antibodies. A T cell epitope is the basic element, orsmallest unit of recognition by a T cell receptor, where the epitopecomprises amino acids essential to receptor recognition (e.g.,approximately 6 or 7 amino acid residues). Amino acid sequences whichmimic those of the T cell epitopes are within the scope of thisinvention.

Screening immunogenic components can be accomplished using one or moreof several different assays. For example, in vitro, peptide T cellstimulatory activity is assayed by contacting a peptide known orsuspected of being immunogenic with an antigen presenting cell whichpresents appropriate MHC molecules in a T cell culture. Presentation ofan immunogenic S. pneumoniae peptide in association with appropriate MHCmolecules to T cells in conjunction with the necessary co-stimulationhas the effect of transmitting a signal to the T cell that induces theproduction of increased levels of cytokines, particularly ofinterleukin-2 and interleukin-4. The culture supernatant can be obtainedand assayed for interleukin-2 or other known cytokines. For example, anyone of several conventional assays for interleukin-2 can be employed,such as the assay described in Proc. Natl. Acad. Sci. USA, 86: 1333(1989) the pertinent portions of which are incorporated herein byreference. A kit for an assay for the production of interferon is alsoavailable from Genzyme Corporation (Cambridge, Mass.).

Alternatively, a common assay for T cell proliferation entails measuringtritiated thymidine incorporation. The proliferation of T cells can bemeasured in vitro by determining the amount of ³H-labeled thymidineincorporated into the replicating DNA of cultured cells. Therefore, therate of DNA synthesis and, in turn, the rate of cell division can bequantified.

Vaccine compositions of the invention containing immunogenic components(e.g., S. pneumoniae polypeptide or fragment thereof or nucleic acidencoding an S. pneumoniae polypeptide or fragment thereof) preferablyinclude a pharmaceutically acceptable carrier. The term“pharmaceutically acceptable carrier” refers to a carrier that does notcause an allergic reaction or other untoward effect in patients to whomit is administered. Suitable pharmaceutically acceptable carriersinclude, for example, one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. Pharmaceutically acceptable carriers may furthercomprise minor amounts of auxiliary substances such as wetting oremulsifying agents, preservatives or buffers, which enhance the shelflife or effectiveness of the antibody. For vaccines of the inventioncontaining S. pneumoniae polypeptides, the polypeptide isco-administered with a suitable adjuvant.

It will be apparent to those of skill in the art that thetherapeutically effective amount of DNA or protein of this inventionwill depend, inter alia, upon the administration schedule, the unit doseof antibody administered, whether the protein or DNA is administered incombination with other therapeutic agents, the immune status and healthof the patient, and the therapeutic activity of the particular proteinor DNA.

Vaccine compositions are conventionally administered parenterally, e.g.,by injection, either subcutaneously or intramuscularly. Methods forintramuscular immunization are described by Wolff et al. (1990) Science247: 1465-1468 and by Sedegah et al. (1994) Immunology 91: 9866-9870.Other modes of administration include oral and pulmonary formulations,suppositories, and transdermal applications. Oral immunization ispreferred over parenteral methods for inducing protection againstinfection by S. pneumoniae. Cain et. al. (1993) Vaccine 11: 637-642.Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate, and thelike.

The vaccine compositions of the invention can include an adjuvant,including, but not limited to aluminum hydroxide;N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP);N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP);N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphos-phoryloxy)-ethylamine(CGP 19835A, referred to a MTP-PE); RIBI, which contains threecomponents from bacteria; monophosphoryl lipid A; trehalose dimycoloate;cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion; andcholera toxin. Others which may be used are non-toxic derivatives ofcholera toxin, including its B subunit, and/or conjugates or geneticallyengineered fusions of the S. pneumoniae polypeptide with cholera toxinor its B subunit, procholeragenoid, fungal polysaccharides, includingschizophyllan, muramyl dipeptide, muramyl dipeptide derivatives, phorbolesters, labile toxin of E. coli, non-S. pneumoniae bacterial lysates,block polymers or saponins.

Other suitable delivery methods include biodegradable microcapsules orimmuno-stimulating complexes (ISCOMs), cochleates, or liposomes,genetically engineered attenuated live vectors such as viruses orbacteria, and recombinant (chimeric) virus-like particles, e.g.,bluetongue. The amount of adjuvant employed will depend on the type ofadjuvant used. For example, when the mucosal adjuvant is cholera toxin,it is suitably used in an amount of 5 mg to 50 mg, for example 10 mg to35 mg. When used in the form of microcapsules, the amount used willdepend on the amount employed in the matrix of the microcapsule toachieve the desired dosage. The determination of this amount is withinthe skill of a person of ordinary skill in the art.

Carrier systems in humans may include enteric release capsulesprotecting the antigen from the acidic environment of the stomach, andincluding S. pneumoniae polypeptide in an insoluble form as fusionproteins. Suitable carriers for the vaccines of the invention areenteric coated capsules and polylactide-glycolide microspheres. Suitablediluents are 0.2 N NaHCO3 and/or saline.

Vaccines of the invention can be administered as a primary prophylacticagent in adults or in children, as a secondary prevention, aftersuccessful eradication of S. pneumoniae in an infected host, or as atherapeutic agent in the aim to induce an immune response in asusceptible host to prevent infection by S. pneumoniae. The vaccines ofthe invention are administered in amounts readily determined by personsof ordinary skill in the art. Thus, for adults a suitable dosage will bein the range of 10 mg to 10 g, preferably 10 mg to 100 mg. A suitabledosage for adults will also be in the range of 5 mg to 500 mg. Similardosage ranges will be applicable for children. Those skilled in the artwill recognize that the optimal dose may be more or less depending uponthe patient's body weight, disease, the route of administration, andother factors. Those skilled in the art will also recognize thatappropriate dosage levels can be obtained based on results with knownoral vaccines such as, for example, a vaccine based on an E. coli lysate(6 mg dose daily up to total of 540 mg) and with an enterotoxigenic E.coli purified antigen (4 doses of 1 mg) (Schulman et al., J. Urol.150:917-921 (1993); Boedecker et al., American GastroenterologicalAssoc. 999:A-222 (1993)). The number of doses will depend upon thedisease, the formulation, and efficacy data from clinical trials.Without intending any limitation as to the course of treatment, thetreatment can be administered over 3 to 8 doses for a primaryimmunization schedule over 1 month (Boedeker, AmericanGastroenterological Assoc. 888:A-222 (1993)).

In a preferred embodiment, a vaccine composition of the invention can bebased on a killed whole E. coli preparation with an immunogenic fragmentof an S. pneumoniae protein of the invention expressed on its surface orit can be based on an E. coli lysate, wherein the killed E. coli acts asa carrier or an adjuvant.

It will be apparent to those skilled in the art that some of the vaccinecompositions of the invention are useful only for preventing S.pneumoniae infection, some are useful only for treating S. pneumoniaeinfection, and some are useful for both preventing and treating S.pneumoniae infection. In a preferred embodiment, the vaccine compositionof the invention provides protection against S. pneumoniae infection bystimulating humoral and/or cell-mediated immunity against S. pneumoniae.It should be understood that amelioration of any of the symptoms of S.pneumoniae infection is a desirable clinical goal, including a lesseningof the dosage of medication used to treat S. pneumoniae-caused disease,or an increase in the production of antibodies in the serum or mucous ofpatients.

Antibodies Reactive with S. pneumoniae Polypeptides

The invention also includes antibodies specifically reactive with thesubject S. pneumoniae polypeptide. Anti-protein/anti-peptide antisera ormonoclonal antibodies can be made by standard protocols (See, forexample, Antibodies: A Laboratory Manual ed. by Harlow and Lane (ColdSpring Harbor Press: 1988)). A mammal such as a mouse, a hamster orrabbit can be immunized with an immunogenic form of the peptide.Techniques for conferring immunogenicity on a protein or peptide includeconjugation to carriers or other techniques well known in the art. Animmunogenic portion of the subject S. pneumoniae polypeptide can beadministered in the presence of adjuvant. The progress of immunizationcan be monitored by detection of antibody titers in plasma or serum.Standard ELISA or other immunoassays can be used with the immunogen asantigen to assess the levels of antibodies.

In a preferred embodiment, the subject antibodies are immunospecific forantigenic determinants of the S. pneumoniae polypeptides of theinvention, e.g. antigenic determinants of a polypeptide of the inventioncontained in the Sequence Listing, or a closely related human ornon-human mammalian homolog (e.g., 90% homologous, more preferably atleast 95% homologous). In yet a further preferred embodiment of theinvention, the anti-S. pneumoniae antibodies do not substantially crossreact (i.e., react specifically) with a protein which is for example,less than 80% percent homologous to a sequence of the inventioncontained in the Sequence Listing. By “not substantially cross react”,it is meant that the antibody has a binding affinity for anon-homologous protein which is less than 10 percent, more preferablyless than 5 percent, and even more preferably less than 1 percent, ofthe binding affinity for a protein of the invention contained in theSequence Listing. In a most preferred embodiment, there is nocross-reactivity between bacterial and mammalian antigens.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with S. pneumoniaepolypeptides. Antibodies can be fragmented using conventional techniquesand the fragments screened for utility in the same manner as describedabove for whole antibodies. For example, F(ab′)₂ fragments can begenerated by treating antibody with pepsin. The resulting F(ab′)₂fragment can be treated to reduce disulfide bridges to produce Fab′fragments. The antibody of the invention is further intended to includebispecific and chimeric molecules having an anti-S. pneumoniae portion.

Both monoclonal and polyclonal antibodies (Ab) directed against S.pneumoniae polypeptides or S. pneumoniae polypeptide variants, andantibody fragments such as Fab′ and F(ab′)₂, can be used to block theaction of S. pneumoniae polypeptide and allow the study of the role of aparticular S. pneumoniae polypeptide of the invention in aberrant orunwanted intracellular signaling, as well as the normal cellularfunction of the S. pneumoniae and by microinjection of anti-S.pneumoniae polypeptide antibodies of the present invention.

Antibodies which specifically bind S. pneumoniae epitopes can also beused in immunohistochemical staining of tissue samples in order toevaluate the abundance and pattern of expression of S. pneumoniaeantigens. Anti S. pneumoniae polypeptide antibodies can be useddiagnostically in immuno-precipitation and immuno-blotting to detect andevaluate S. pneumoniae levels in tissue or bodily fluid as part of aclinical testing procedure. Likewise, the ability to monitor S.pneumoniae polypeptide levels in an individual can allow determinationof the efficacy of a given treatment regimen for an individual afflictedwith such a disorder. The level of an S. pneumoniae polypeptide can bemeasured in cells found in bodily fluid, such as in urine samples or canbe measured in tissue, such as produced by gastric biopsy. Diagnosticassays using anti-S. pneumoniae antibodies can include, for example,immunoassays designed to aid in early diagnosis of S. pneumoniaeinfections. The present invention can also be used as a method ofdetecting antibodies contained in samples from individuals infected bythis bacterium using specific S. pneumoniae antigens.

Another application of anti-S. pneumoniae polypeptide antibodies of theinvention is in the immunological screening of cDNA librariesconstructed in expression vectors such as lgt11, lgt18-23, IZAP, andIORF8. Messenger libraries of this type, having coding sequencesinserted in the correct reading frame and orientation, can producefusion proteins. For instance, lgt11 will produce fusion proteins whoseamino termini consist of β-galactosidase amino acid sequences and whosecarboxy termini consist of a foreign polypeptide. Antigenic epitopes ofa subject S. pneumoniae polypeptide can then be detected withantibodies, as, for example, reacting nitrocellulose filters lifted frominfected plates with anti-S. pneumoniae polypeptide antibodies. Phage,scored by this assay, can then be isolated from the infected plate.Thus, the presence of S. pneumoniae gene homologs can be detected andcloned from other species, and alternate isoforms (including splicingvariants) can be detected and cloned.

Kits Containing Nucleic Acids, Polypeptides or Antibodies of theInvention

The nucleic acid, polypeptides and antibodies of the invention can becombined with other reagents and articles to form kits. Kits fordiagnostic purposes typically comprise the nucleic acid, polypeptides orantibodies in vials or other suitable vessels. Kits typically compriseother reagents for performing hybridization reactions, polymerase chainreactions (PCR), or for reconstitution of lyophilized components, suchas aqueous media, salts, buffers, and the like. Kits may also comprisereagents for sample processing such as detergents, chaotropic salts andthe like. Kits may also comprise immobilization means such as particles,supports, wells, dipsticks and the like. Kits may also comprise labelingmeans such as dyes, developing reagents, radioisotopes, fluorescentagents, luminescent or chemiluminescent agents, enzymes, intercalatingagents and the like. With the nucleic acid and amino acid sequenceinformation provided herein, individuals skilled in art can readilyassemble kits to serve their particular purpose. Kits further caninclude instructions for use.

Bio Chips and Microarrays

The nucleic acid sequence of the present invention may be used to detectS. pneumoniae or other species of Streptococcus acid sequence using biochip technology. Bio chips containing arrays of nucleic acid sequencecan also be used to measure expression of genes of S. pneumoniae orother species of Streptococcus. For example, to diagnose a patient witha S. pneumoniae or other Streptococcus infection, a sample from a humanor animal can be used as a probe on a bio chip containing an array ofnucleic acid sequence from the present invention. In addition, a samplefrom a disease state can be compared to a sample from a non-diseasestate which would help identify a gene that is up-regulated or expressedin the disease state. This would provide valuable insight as to themechanism by which the disease manifests. Changes in gene expression canalso be used to identify critical pathways involved in drug transport ormetabolism, and may enable the identification of novel targets involvedin virulence or host cell interactions involved in maintenance of aninfection. Procedures using such techniques have been described by Brownet al., 1995, Science 270: 467-470.

Bio chips can also be used to monitor the genetic changes of potentialtherapeutic compounds including, deletions, insertions or mismatches.Once the therapeutic is added to the patient, changes to the geneticsequence can be evaluated for its efficacy. In addition, the nucleicacid sequence of the present invention can be used to determineessential genes in cell cycling. As described in Iyer et al., 1999(Science, 283:83-87) genes essential in the cell cycle can be identifiedusing bio chips. Furthermore, the present invention provides nucleicacid sequence which can be used with bio chip technology to understandregulatory networks in bacteria, measure the response to environmentalsignals or drugs as in drug screening, and study virulence induction.(Mons et al., 1998, Nature Biotechnology, 16: 45-48. Patents teachingthis technology include U.S. Pat. Nos. 5,445,934, 5,744,305, and5800992.

Drug Screening Assays Using S. pneumoniae Polypeptides

By making available purified and recombinant S. pneumoniae polypeptides,the present invention provides assays which can be used to screen fordrugs which are either agonists or antagonists of the normal cellularfunction, in this case, of the subject S. pneumoniae polypeptides, or oftheir role in intracellular signaling. Such inhibitors or potentiatorsmay be useful as new therapeutic agents to combat S. pneumoniaeinfections in humans. A variety of assay formats will suffice and, inlight of the present inventions, will be comprehended by the skilledartisan.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity with other proteinsor change in enzymatic properties of the molecular target. Accordingly,in an exemplary screening assay of the present invention, the compoundof interest is contacted with an isolated and purified S. pneumoniaepolypeptide.

Screening assays can be constructed in vitro with a purified S.pneumoniae polypeptide or fragment thereof, such as an S. pneumoniaepolypeptide having enzymatic activity, such that the activity of thepolypeptide produces a detectable reaction product. The efficacy of thecompound can be assessed by generating dose response curves from dataobtained using various concentrations of the test compound. Moreover, acontrol assay can also be performed to provide a baseline forcomparison. Suitable products include those with distinctive absorption,fluorescence, or chemiluminescence properties, for example, becausedetection may be easily automated. A variety of synthetic or naturallyoccurring compounds can be tested in the assay to identify those whichinhibit or potentiate the activity of the S. pneumoniae polypeptide.Some of these active compounds may directly, or with chemicalalterations to promote membrane permeability or solubility, also inhibitor potentiate the same activity (e.g., enzymatic activity) in whole,live S. pneumoniae cells.

Overexpression Assays

Overexpression assays are based on the premise that overproduction of aprotein would lead to a higher level of resistance to compounds thatselectively interfere with the function of that protein. Overexpressionassays may be used to identify compounds that interfere with thefunction of virtually any type of protein, including without limitationenzymes, receptors, DNA- or RNA-binding proteins, or any proteins thatare directly or indirectly involved in regulating cell growth.

Typically, two bacterial strains are constructed. One contains a singlecopy of the gene of interest, and a second contains several copies ofthe same gene. Identification of useful inhibitory compounds of thistype of assay is based on a comparison of the activity of a testcompound in inhibiting growth and/or viability of the two strains. Themethod involves constructing a nucleic acid vector that directs highlevel expression of a particular target nucleic acid. The vectors arethen transformed into host cells in single or multiple copies to producestrains that express low to moderate and high levels of protein encodingby the target sequence (strain A and B, respectively). Nucleic acidcomprising sequences encoding the target gene can, of course, bedirectly integrated into the host cell.

Large numbers of compounds (or crude substances which may contain activecompounds) are screened for their effect on the growth of the twostrains. Agents which interfere with an unrelated target equally inhibitthe growth of both strains. Agents which interfere with the function ofthe target at high concentration should inhibit the growth of bothstrains. It should be possible, however, to titrate out the inhibitoryeffect of the compound in the overexpressing strain. That is, if thecompound is affecting the particular target that is being tested, itshould be possible to inhibit the growth of strain A at a concentrationof the compound that allows strain B to grow.

Alternatively, a bacterial strain is constructed that contains the geneof interest under the control of an inducible promoter. Identificationof useful inhibitory agents using this type of assay is based on acomparison of the activity of a test compound in inhibiting growthand/or viability of this strain under both inducing and non-inducingconditions. The method involves constructing a nucleic acid vector thatdirects high-level expression of a particular target nucleic acid. Thevector is then transformed into host cells that are grown under bothnon-inducing and inducing conditions (conditions A and B, respectively).

Large numbers of compounds (or crude substances which may contain activecompounds) are screened for their effect on growth under these twoconditions. Agents that interfere with the function of the target shouldinhibit growth under both conditions. It should be possible, however, totitrate out the inhibitory effect of the compound in the overexpressingstrain. That is, if the compound is affecting the particular target thatis being tested, it should be possible to inhibit growth under conditionA at a concentration that allows the strain to grow under condition B.

Ligand-Binding Assays

Many of the targets according to the invention have functions that havenot yet been identified. Ligand-binding assays are useful to identifyinhibitor compounds that interfere with the function of a particulartarget, even when that function is unknown. These assays are designed todetect binding of test compounds to particular targets. The detectionmay involve direct measurement of binding. Alternatively, indirectindications of binding may involve stabilization of protein structure ordisruption of a biological function. Non-limiting examples of usefulligand-binding assays are detailed below.

A useful method for the detection and isolation of binding proteins isthe Biomolecular Interaction Assay (BIAcore) system developed byPharmacia Biosensor and described in the manufacturer's protocol (LKBPharmacia, Sweden). The BIAcore system uses an affinity purifiedanti-GST antibody to immobilize GST-fusion proteins onto a sensor chip.The sensor utilizes surface plasmon resonance which is an opticalphenomenon that detects changes in refractive indices. In accordancewith the practice of the invention, a protein of interest is coated ontoa chip and test compounds are passed over the chip. Binding is detectedby a change in the refractive index (surface plasmon resonance).

A different type of ligand-binding assay involves scintillationproximity assays (SPA, described in U.S. Pat. No. 4,568,649).

Another type of ligand binding assay, also undergoing development, isbased on the fact that proteins containing mitochondrial targetingsignals are imported into isolated mitochondria in vitro (Hurt et al.,1985, Embo J. 4:2061-2068; Eilers and Schatz, Nature, 1986,322:228-231). In a mitochondrial import assay, expression vectors areconstructed in which nucleic acids encoding particular target proteinsare inserted downstream of sequences encoding mitochondrial importsignals. The chimeric proteins are synthesized and tested for theirability to be imported into isolated mitochondria in the absence andpresence of test compounds. A test compound that binds to the targetprotein should inhibit its uptake into isolated mitochondria in vitro.

Another ligand-binding assay is the yeast two-hybrid system (Fields andSong, 1989, Nature 340:245-246). The yeast two-hybrid system takesadvantage of the properties of the GAL4 protein of the yeastSaccharomyces cerevisiae. The GAL4 protein is a transcriptionalactivator required for the expression of genes encoding enzymes ofgalactose utilization. This protein consists of two separable andfunctionally essential domains: an N-terminal domain which binds tospecific DNA sequences (UAS_(G)); and a C-terminal domain containingacidic regions, which is necessary to activate transcription. The nativeGAL4 protein, containing both domains, is a potent activator oftranscription when yeast are grown on galactose media. The N-terminaldomain binds to DNA in a sequence-specific manner but is unable toactivate transcription. The C-terminal domain contains the activatingregions but cannot activate transcription because it fails to belocalized to UAS_(G). In the two-hybrid system, a system of two hybridproteins containing parts of GAL4: (1) a GAL4 DNA-binding domain fusedto a protein ‘X’ and (2) a GAL4 activation region fused to a protein‘Y’. If X and Y can form a protein-protein complex and reconstituteproximity of the GAL4 domains, transcription of a gene regulated byUAS_(G) occurs. Creation of two hybrid proteins, each containing one ofthe interacting proteins X and Y, allows the activation region ofUAS_(G) to be brought to its normal site of action.

The binding assay described in Fodor et al., 1991, Science 251:767-773,which involves testing the binding affinity of test compounds for aplurality of defined polymers synthesized on a solid substrate, may alsobe useful.

Compounds which bind to the polypeptides of the invention arepotentially useful as antibacterial agents for use in therapeuticcompositions.

Pharmaceutical formulations suitable for antibacterial therapy comprisethe antibacterial agent in conjunction with one or more biologicallyacceptable carriers. Suitable biologically acceptable carriers include,but are not limited to, phosphate-buffered saline, saline, deionizedwater, or the like. Preferred biologically acceptable carriers arephysiologically or pharmaceutically acceptable carriers.

The antibacterial compositions include an antibacterial effective amountof active agent. Antibacterial effective amounts are those quantities ofthe antibacterial agents of the present invention that affordprophylactic protection against bacterial infections or which result inamelioration or cure of an existing bacterial infection. Thisantibacterial effective amount will depend upon the agent, the locationand nature of the infection, and the particular host. The amount can bedetermined by experimentation known in the art, such as by establishinga matrix of dosages and frequencies and comparing a group ofexperimental units or subjects to each point in the matrix.

The antibacterial active agents or compositions can be formed intodosage unit forms, such as for example, creams, ointments, lotions,powders, liquids, tablets, capsules, suppositories, sprays, aerosols orthe like. If the antibacterial composition is formulated into a dosageunit form, the dosage unit form may contain an antibacterial effectiveamount of active agent. Alternatively, the dosage unit form may includeless than such an amount if multiple dosage unit forms or multipledosages are to be used to administer a total dosage of the active agent.Dosage unit forms can include, in addition, one or more excipient(s),diluent(s), disintegrant(s), lubricant(s), plasticizer(s), colorant(s),dosage vehicle(s), absorption enhancer(s), stabilizer(s),bactericide(s), or the like.

For general information concerning formulations, see, e.g., Gilman etal. (eds.), 1990, Goodman and Gilman's: The Pharmacological Basis ofTherapeutics, 8th ed., Pergamon Press; and Remington's PharmaceuticalSciences, 17th ed., 1990, Mack Publishing Co., Easton, Pa.; Avis et al.(eds.), 1993, Pharmaceutical Dosage Forms: Parenteral Medications,Dekker, New York; Lieberman et al (eds.), 1990, Pharmaceutical DosageForms: Disperse Systems, Dekker, New York.

The antibacterial agents and compositions of the present invention areuseful for preventing or treating S. pneumoniae infections. Infectionprevention methods incorporate a prophylactically effective amount of anantibacterial agent or composition. A prophylactically effective amountis an amount effective to prevent S. pneumoniae infection and willdepend upon the specific bacterial strain, the agent, and the host.These amounts can be determined experimentally by methods known in theart and as described above.

S. pneumoniae infection treatment methods incorporate a therapeuticallyeffective amount of an antibacterial agent or composition. Atherapeutically effective amount is an amount sufficient to ameliorateor eliminate the infection. The prophylactically and/or therapeuticallyeffective amounts can be administered in one administration or overrepeated administrations. Therapeutic administration can be followed byprophylactic administration, once the initial bacterial infection hasbeen resolved.

The antibacterial agents and compositions can be administered topicallyor systemically. Topical application is typically achieved byadministration of creams, ointments, lotions, or sprays as describedabove. Systemic administration includes both oral and parental routes.Parental routes include, without limitation, subcutaneous,intramuscular, intraperitoneal, intravenous, transdermal, inhalation andintranasal administration.

EXEMPLIFICATION

I. Cloning and Sequencing of S. pneumoniae DNA

S. pneumoniae chromosomal DNA was isolated according to a basic DNAprotocol outlined in Schleif R. F. and Wensink P. C., Practical Methodsin Molecular Biology, p. 98, Springer-Verlag, NY., 1981, with minormodifications. Briefly, cells were pelleted, resuspended in TE (10 mMTris, 1 mM EDTA, pH 7.6) and GES lysis buffer (5.1 M guanidiumthiocyanate, 0.1 M EDTA, pH 8.0, 0.5% N-laurylsarcosine) was added.Suspension was chilled and ammonium acetate (NH4Ac) was added to finalconcentration of 2.0 M. DNA was extracted, first with chloroform, thenwith phenol-chloroform, and reextracted with chloroform. DNA wasprecipitated with isopropanol, washed twice with 70% EtOH, dried andresuspended in TE.

Following isolation whole genomic S. pneumoniae DNA was nebulized(Bodenteich et al., Automated DNA Sequencing and Analysis (J. C. Venter,ed.), Academic Press, 1994) to a median size of 2000 bp. Afternebulization, the DNA was concentrated and separated on a standard 1%agarose gel. Several fractions, corresponding to approximate sizes1000-1500 bp, 1500-2000 bp, 2000-2500 bp, 2500-3000 bp, were excisedfrom the gel and purified by the GeneClean procedure (Bio101, Inc.).

The purified DNA fragments were then blunt-ended using T4 DNApolymerase. The healed DNA was then ligated to unique BstXI-linkeradapters (5′ GTCTTCACCACGGGG (SEQ ID NO: 5323) and 5′ GTGGTGAAGAC (SEQID NO: 5324) in 100-1000 fold molar excess). These linkers arecomplimentary to the BstXI-cut pMPX vectors, while the overhang is notself-complimentary. Therefore, the linkers will not concatemerize norwill the cut-vector religate itself easily. The linker-adopted insertswere separated from the unincorporated linkers on a 1% agarose gel andpurified using GeneClean. The linker-adopted inserts were then ligatedto each of 20 pMPX vectors to construct a series of “shotgun” subclonelibraries. Blunt ended vector was used for cloning into the PUC19vector. The vectors contain an out-of-frame lacZ gene at the cloningsite which becomes in-frame in the event that an adapter-dimer iscloned, allowing these to be avoided by their blue-color.

All subsequent steps were based either on the multiplex DNA sequencingprotocols outlined in Church G. M. and Kieffer-Higgins S., Science240:185-188, 1988 or by ABI377 automated DNA sequencing methods. Onlymajor modifications to the protocols are highlighted. Briefly, each ofthe 20 vectors was then transformed into DH5a competent cells(Gibco/BRL, DH5a transformation protocol). The libraries were assessedby plating onto antibiotic plates containing ampicillin, methicillin andIPTG/Xgal. The plates were incubated overnight at 37° C. Successfultransformants were then used for plating of clones and pooling into themultiplex pools. The clones were picked and pooled into 40 ml growthmedium cultures. The cultures were grown overnight at 37° C. DNA waspurified using the Qiagen Midi-prep kits and Tip-100 columns (Qiagen,Inc.). In this manner, 100 mg of DNA was obtained per pool.

These purified DNA samples were then sequenced either using themultiplex DNA sequencing based on chemical degradation methods (ChurchG. M. and Kieffer-Higgins S., Science 240:185-188, 1988) or bySequithrem (Epicenter Technologies) dideoxy sequencing protocols or byABI dye-terminator chemistry. For the multiplex portion the sequencingreactions were electrophoresed and transferred onto nylon membranes bydirect transfer electrophoresis from 40 cm gels (Richterich P. andChurch G. M., Methods in Enzymology 218:187-222, 1993). The DNA wascovalently bound to the membranes by exposure to ultraviolet light, andhybridized with labeled oligonucleotides complimentary to tag sequenceson the vectors (Church, supra). The membranes were washed to rinse offnon-specifically bound probe, and exposed to X-ray film to visualizeindividual sequence ladders. After autoradiography, the hybridized probewas removed by incubation at 65° C., and the hybridization cyclerepeated with another tag sequence until the membrane had been probed 41times. Thus, each gel produced a large number of films, each containingnew sequencing information. Whenever a new blot was processed, it wasinitially probed for an internal standard sequence added to each of thepools. Digital images of the films were generated using a laser-scanningdensitometer (Molecular Dynamics, Sunnyvale, Calif.). The digitizedimages were processed on computer workstations (VaxStation 4000's) usingthe program REPLICA™ (Church et al., Automated DNA Sequencing andAnalysis (J. C. Venter, ed.), Academic Press, 1994). Image processingincluded lane straightening, contrast adjustment to smooth out intensitydifferences, and resolution enhancement by iterative gaussiandeconvolution. The sequences were then converted to an SCF format sothat processing and assembly could proceed on UNIX machines. The ABI dyeterminator sequence reads were run on ABI377 machines and the data wasdirectly transferred to UNIX machines following lane tracking of thegels. All multiplex and ABI reads were assembled using PHRAP (P. Green,Abstracts of DOE Human Genome Program Contractor-Grantee Workshop V,January 1996, p. 157) with default parameters and not using qualityscores. The initial assembly was done at 7 fold coverage and yielded 511contigs. Short read length fragments of 200 bp or less found on the endsof contigs facing in the appropriate direction were used to extend offthe end of the contigs. These reads were then resequenced with primersusing ABI technology to give sequences with a read length of 500 or morebases. This allowed end extensions to be performed without ordering newprimers. In addition, missing mates (sequences from clones that onlygave one strand reads) were identified and sequenced with ABI technologyto allow the identification of additional overlapping contigs.

End-sequencing of randomly picked genomic lambda was also performed.Sequencing on a both sides was done for all lambda sequences. The lambdalibrary backbone helped to verify the integrity of the assembly andallowed closure of some of the physical gaps.

To identify S. pneumoniae polypeptides the complete genomic sequence ofS. pneumoniae were analyzed essentially as follows: First, all possiblestop-to-stop open reading frames (ORFs) greater than 180 nucleotides inall six reading frames were translated into amino acid sequences.Second, the identified ORFs were analyzed for homology to known(archeabacter, prokaryotic and eukaryotic) protein sequences. Third, thepredicted coding regions of the sequences and start codons wereevaluated with the programs GENEMARK™ (Borodovsky and McIninch, 1993,Comp. Chem. 17:123) and Glimmer (Fraser et al, Nature, 1997).

Identification, Cloning and Expression of S. pneumoniae Nucleic Acids

Expression and purification of the S. pneumoniae polypeptides of theinvention can be performed essentially as outlined below.

To facilitate the cloning, expression and purification of membrane andsecreted proteins from S. pneumoniae, a gene expression system, such asthe pET System (Novagen), for cloning and expression of recombinantproteins in E. coli, is selected. Also, a DNA sequence encoding apeptide tag, the His-Tag, is fused to the 3′ end of DNA sequences ofinterest in order to facilitate purification of the recombinant proteinproducts. The 3′ end is selected for fusion in order to avoid alterationof any 5′ terminal signal sequence.

PCR Amplification and Cloning of Nucleic Acids Containing ORF's EncodingEnzymes

Nucleic acids chosen (for example, from the nucleic acids set forth inSEQ ID NO: 1-SEQ ID NO: 2661) for cloning from the 14453 strain of S.pneumoniae are prepared for amplification cloning by polymerase chainreaction (PCR). Synthetic oligonucleotide primers specific for the 5′and 3′ ends of open reading frames (ORFs) are designed and purchasedfrom GibcoBRL Life Technologies (Gaithersburg, Md., USA). All forwardprimers (specific for the 5′ end of the sequence) are designed toinclude an NcoI cloning site at the extreme 5′ terminus. These primersare designed to permit initiation of protein translation at a methionineresidue followed by a valine residue and the coding sequence for theremainder of the native S. pneumoniae DNA sequence. All reverse primers(specific for the 3′ end of any S. pneumoniae ORF) include a EcoRI siteat the extreme 5′ terminus to permit cloning of each S. pneumoniaesequence into the reading frame of the pET-28b. The pET-28b vectorprovides sequence encoding an additional 20 carboxy-terminal amino acidsincluding six histidine residues (at the extreme C-terminus), whichcomprise the His-Tag.

Genomic DNA prepared from strain 14453 of S. pneumoniae is used as thesource of template DNA for PCR amplification reactions (CurrentProtocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel etal., eds., 1994). To amplify a DNA sequence containing an S. pneumoniaeORF, genomic DNA (50 nanograms) is introduced into a reaction vialcontaining 2 mM MgCl₂, 1 micromolar synthetic oligonucleotide primers(forward and reverse primers) complementary to and flanking a defined S.pneumoniae ORF, 0.2 mM of each deoxynucleotide triphosphate; dATP, dGTP,dCTP, dTTP and 2.5 units of heat stable DNA polymerase (Amplitaq, RocheMolecular Systems, Inc., Branchburg, N.J., USA) in a final volume of 100microliters.

Upon completion of thermal cycling reactions, each sample of amplifiedDNA is washed and purified using the Qiaquick Spin PCR purification kit(Qiagen, Gaithersburg, Md., USA). All amplified DNA samples aresubjected to digestion with the restriction endonucleases, e.g., NcoIand EcoRI (New England BioLabs, Beverly, Mass., USA) (Current Protocolsin Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al.,eds., 1994). DNA samples are then subjected to electrophoresis on 1.0%NuSeive (FMC BioProducts, Rockland, Me. USA) agarose gels. DNA isvisualized by exposure to ethidium bromide and long wave uv irradiation.DNA contained in slices isolated from the agarose gel is purified usingthe Bio 101 GeneClean Kit protocol (Bio 101 Vista, Calif., USA).

Cloning of S. pneumoniae Nucleic Acids into an Expression Vector

The pET-28b vector is prepared for cloning by digestion withendonucleases, e.g., NcoI and EcoRI (Current Protocols in MolecularBiology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). ThepET-28a vector, which encodes a His-Tag that can be fused to the 5, endof an inserted gene, is prepared by digestion with appropriaterestriction endonucleases.

Following digestion, DNA inserts are cloned (Current Protocols inMolecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds.,1994) into the previously digested pET-28b expression vector. Productsof the ligation reaction are then used to transform the BL21 strain ofE. coli (Current Protocols in Molecular Biology, John Wiley and Sons,Inc., F. Ausubel et al., eds., 1994) as described below.

Transformation of Competent Bacteria with Recombinant Plasmids

Competent bacteria, E coli strain BL21 or E. coli strain BL21 (DE3), aretransformed with recombinant pET expression plasmids carrying the clonedS. pneumoniae sequences according to standard methods (Current Protocolsin Molecular, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994).Briefly, 1 microliter of ligation reaction is mixed with 50 microlitersof electrocompetent cells and subjected to a high voltage pulse, afterwhich, samples are incubated in 0.45 milliliters SOC medium (0.5% yeastextract, 2.0% tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4and 20, mM glucose) at 37° C. with shaking for 1 hour. Samples are thenspread on LB agar plates containing 25 microgram/ml kanamycin sulfatefor growth overnight. Transformed colonies of BL21 are then picked andanalyzed to evaluate cloned inserts as described below.

Identification of Recombinant Expression Vectors with S. pneumoniaeNucleic Acids

Individual BL21 clones transformed with recombinant pET-28b S.pneumoniae ORFs are analyzed by PCR amplification of the cloned insertsusing the same forward and reverse primers, specific for each S.pneumoniae sequence, that were used in the original PCR amplificationcloning reactions. Successful amplification verifies the integration ofthe S. pneumoniae sequences in the expression vector (Current Protocolsin Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al.,eds., 1994).

Isolation and Preparation of Nucleic Acids from Transformants

Individual clones of recombinant pET-28b vectors carrying properlycloned S. pneumoniae ORFs are picked and incubated in 5 mls of LB brothplus 25 microgram/ml kanamycin sulfate overnight. The following dayplasmid DNA is isolated and purified using the Qiagen plasmidpurification protocol (Qiagen Inc., Chatsworth, Calif., USA).

Expression Of Recombinant S. pneumoniae Sequences In E. coli

The pET vector can be propagated in any E. coli K-12 strain e.g. HMS174,HB101, JM109, DH5, etc. for the purpose of cloning or plasmidpreparation. Hosts for expression include E. coli strains containing achromosomal copy of the gene for T7 RNA polymerase. These hosts arelysogens of bacteriophage DE3, a lambda derivative that carries the lacIgene, the lacUV5 promoter and the gene for T7 RNA polymerase. T7 RNApolymerase is induced by addition of isopropyl-B-D-thiogalactoside(IPTG), and the T7 RNA polymerase transcribes any target plasmid, suchas pET-28b, carrying its gene of interest. Strains used include: BL21(DE3) (Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J.W. (1990) Meth. Enzymol. 185, 60-89).

To express recombinant S. pneumoniae sequences, 50 nanograms of plasmidDNA isolated as described above is used to transform competent BL21(DE3) bacteria as described above (provided by Novagen as part of thepET expression system kit). The lacZ gene (beta-galactosidase) isexpressed in the pET-System as described for the S. pneumoniaerecombinant constructions. Transformed cells are cultured in SOC mediumfor 1 hour, and the culture is then plated on LB plates containing 25micrograms/ml kanamycin sulfate. The following day, bacterial coloniesare pooled and grown in LB medium containing kanamycin sulfate (25micrograms/ml) to an optical density at 600 nM of 0.5 to 1.0 O.D. units,at which point, 1 millimolar IPTG was added to the culture for 3 hoursto induce gene expression of the S. pneumoniae recombinant DNAconstructions.

After induction of gene expression with IPTG, bacteria are pelleted bycentrifugation in a Sorvall RC-3B centrifuge at 3500×g for 15 minutes at4° C. Pellets are resuspended in 50 milliliters of cold 10 mM Tris-HCl,pH 8.0, 0.1 M NaCl and 0.1 mM EDTA (STE buffer). Cells are thencentrifuged at 2000×g for 20 min at 4° C. Wet pellets are weighed andfrozen at −80° C. until ready for protein purification.

A variety of methodologies known in the art can be utilized to purifythe isolated proteins. (Current Protocols in Protein Science, John Wileyand Sons, Inc., J. E. Coligan et al., eds., 1995). For example, thefrozen cells may be thawed, resupended in buffer and ruptured by severalpassages through a small volume microfluidizer (Model M-110S,Microfluidics International Corporation, Newton, Mass.). The resultanthomogenate may be centrifuged to yield a clear supernatant (crudeextract) and following filtration the crude extract may be fractionatedover columns. Fractions may be monitored by absorbance at OD₂₈₀ nm. andpeak fractions may analyzed by SDS-PAGE

The concentrations of purified protein preparations may be quantifiedspectrophotometrically using absorbance coefficients calculated fromamino acid content (Perkins, S. J. 1986 Eur. J. Biochem. 157, 169-180).Protein concentrations are also measured by the method of Bradford, M.M. (1976) Anal. Biochem. 72, 248-254, and Lowry, O. H., Rosebrough, N.,Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, pages 265-275,using bovine serum albumin as a standard.

SDS-polyacrylamide gels of various concentrations may be purchased fromBioRad (Hercules, Calif., USA), and stained with Coomassie blue.Molecular weight markers may include rabbit skeletal muscle myosin (200kDa), E. coli (-galactosidase (116 kDa), rabbit muscle phosphorylase B(97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), bovinecarbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), eggwhite lysozyme (14.4 kDa) and bovine aprotinin (6.5 kDa).

EQUIVALENTS

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. An isolated nucleic acid encoding a S. pneumoniae surface proteinhaving at least 90% identity to SEQ ID NO:
 5226. 2. A recombinantexpression vector comprising the nucleic acid of claim 1 operably linkedto a transcription regulatory element.
 3. A cell comprising therecombinant expression vector of claim
 2. 4. An isolated nucleic acidselected from the group consisting of: a) a nucleic acid encoding a S.pneumoniae surface protein having at least 90% identity to SEQ ID NO:5226; b) a nucleic acid fully complementary to a nucleic acid encoding aS. pneumoniae surface protein having at least 90% identity to SEQ ID NO:5226; and c) an RNA of a) or b), wherein U is substituted for T.
 5. Arecombinant expression vector comprising the nucleic acid of claim 4operably linked to a transcription regulatory element.
 6. A cellcomprising the recombinant expression vector of claim
 5. 7. The isolatednucleic acid of claim 1, wherein the S. pneumoniae surface proteinelicits an immune response.
 8. The isolated nucleic acid of claim 1,wherein the S. pneumoniae surface protein provides protective immunityagainst an infection by S. pneumoniae.
 9. The isolated nucleic acid ofclaim 4, wherein the S. pneumoniae surface protein elicits an immuneresponse.
 10. The isolated nucleic acid of claim 4, wherein the S.pneumoniae surface protein provides protective immunity against aninfection by S. pneumoniae.